Activation of a Neural Brain-Testicular Pathway Rapidly Lowers Leydig Cell Levels of the Steroidogenic Acute Regulatory Protein an
http://www.100md.com
《内分泌学杂志》
The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037
Abstract
Activation of a neural brain-testicular pathway by the intracerebroventricular injection of the -adrenergic agonist isoproterenol (ISO), the hypothalamic peptide corticotropin-releasing factor (CRF), or alcohol (EtOH) rapidly decreases the testosterone (T) response to human chorionic gonadotropin. To elucidate the intratesticular mechanisms responsible for this phenomenon, we investigated the influence of intracerebroventricular-injected ISO, CRF, or EtOH on levels of the steroidogenic acute regulatory (StAR) protein, the peripheral-type benzodiazepine receptor (PBR), and the cytochrome P450 side-chain cleavage enzyme in semipurified Leydig cells. ISO (10 μg), CRF (5 μg), or EtOH (5 μl of 200 proof, a dose that does not induce neuronal damage nor leaks to the periphery) rapidly decreased StAR and PBR but not cytochrome P450 side-chain cleavage enzyme protein levels. Levels of the variant of the neuronal nitric oxide synthase (nNOS) that is restricted to Leydig cells, TnNOS, significantly increased in response to ISO, CRF, and EtOH over the time course of altered StAR/PBR concentrations. However, pretreatment of the rats with Nwnitro-arginine methylester, which blocked ISO-induced increases in TnNOS, neither restored the T response to human chorionic gonadotropin nor prevented the decreases in StAR and PBR. These results provide evidence of concomitant changes in Leydig cell StAR and PBR levels in live rats. They also indicate that activation of a neural brain-testicular pathway rapidly decreases concentrations of these steroidogenic proteins while up-regulating testicular NO production. However, additional studies are necessary to elucidate the functional role played by this gas in our model.
Introduction
TESTOSTERONE (T) SYNTHESIS in the Leydig cells is traditionally thought to be governed by the release of LH from the pituitary, itself under the control of the hypothalamic peptide LHRH (1, 2, 3). However, certain stressors are known to cause a rapid decrease in T release that occurs without measurable changes in mean LH levels (4). The mechanisms by which this inhibition takes place have been the subject of much speculation, including an altered pattern of pulsatile LH secretion as well as the presence in the circulation and/or the testes of compounds that inhibit steroidogenesis (reviewed in Ref.5). We recently proposed an additional and not necessarily mutually exclusive possibility, namely the existence of an inhibitory brain-testes pathway that is activated by stress-related compounds. This hypothesis was based on our observation that the intracerebroventricular (icv) injection of the -adrenergic agonist isoproterenol (ISO), corticotropin-releasing factor (CRF), or alcohol (EtOH) all rapidly and significantly blunted T secretion induced by the exogenous injection of human chorionic gonadotropin (hCG) (4, 6, 7, 8, 9). Blocking LH release with a potent LHRH antagonist neither mimicked nor influenced the suppressive influence of the icv-administered molecules, thereby indicating that this influence was independent of the pituitary. Normal testicular activity was similarly not restored by prior treatment with CRF antibodies, which did not support the hypothesis that the effect of the proposed inhibitory pathway relied on adrenal steroids (5, 6, 7, 8). Finally, we showed that none of the icv treatments we used altered testicular blood flow (9), thereby eliminating the possibility that the blunted T response measured in these experiments was a result of decreased delivery of gonadotropins to and/or impaired T release from Leydig cells. This pathway was subsequently mapped with the retrograde transneuronal tracer pseudo-rabies virus (10, 11, 12) and shown to include specific areas of the hypothalamus (4, 9). However, the testicular mechanisms through which stimulation of this circuit interferes with T synthesis and release have not yet been elucidated. Our earlier studies had indicated that the icv injection of IL-1 did not alter testicular LH/hCG binding affinity but lowered testicular steroidogenic acute regulatory (StAR) protein levels without suppressing mitochondrial function because the addition of a water-permeable form of cholesterol to decapsulated testes prevented the inhibitory influence of the cytokine (13). In the present work, we investigated the possible roles of three proteins, the StAR protein (14, 15), the peripheral-type benzodiazepine receptor (PBR) (16, 17), and the cholesterol side-chain cleavage cytochrome P450 (P450scc), in response to centrally injected compounds known to decrease the T response to hCG.
StAR is synthesized de novo as a 37-kDa precursor that actively binds cholesterol in the cytosol and is then imported into the mitochondria where it is rapidly cleaved to a 30-kDa mature form (18, 19). This highly conserved protein (20) plays an essential role in steroid hormone synthesis by regulating the transfer of cholesterol, which is the substrate for all steroid hormones, from the outer to the inner mitochondrial membrane where P450scc catalyzes its conversion to pregnenolone (3, 21, 22, 23). Delivery of cholesterol to P450scc is thus considered the true rate-limiting step of steroidogenesis. However, StAR is now known to interact with PBR, an 18-kDa protein located on the outer mitochondrial membrane where it is expressed as a cholesterol transporter and serves as a cholesterol channel (24, 25, 26, 27, 28). Specifically, StAR binds cholesterol in the cytoplasm and transfers it to PBR, a step that is followed by transport of this molecule across the outer to the inner mitochondrial membrane (16, 29, 30). The fact that PBR is required for StAR import into mitochondria (31, 32) thus indicates that both proteins represent essential components of steroidogenesis. Indeed, changes in StAR and/or PBR levels have been associated with related changes in the levels of a variety of steroids in the nervous system (33), the adrenals (34, 35, 36, 37), and the corpus luteum (38). However, to our knowledge, there is no information regarding concurrent responses of gonadal StAR and PBR to stressors delivered to the intact animal.
We show here that icv-injected ISO, CRF, or EtOH all rapidly lowered the testicular levels of both StAR and PBR in paradigms associated with blunted T release. These results then led us to ask whether we could also detect changes in the testicular levels of molecules known to inhibit steroidogenesis that might play a functional role in decreased StAR and PBR levels. Our initial focus was on the unstable gas nitric oxide (NO), which has been reportedly found in testes using various detection methods such as nitrite/nitrate levels, citrulline conversion, and NO synthase (NOS) measurement by histochemical procedure (39, 40). Stressors such as immobilization and proinflammatory cytokines, as well as alcohol, were found to increase testicular NO levels (40, 41, 42), and these increases appear to be related to both constitutive and inducible forms of NO (40, 42, 43). Interestingly, a 125-kDa isoform alternate promoter of the 150-kDa human neuronal NOS, the constitutive enzyme responsible for NO formation (44, 45), was recently found in mice Leydig cells and called T neuronal (n)NOS (46, 47). However, despite much evidence that NO interferes with male sex steroid synthesis (41, 48, 49, 50, 51), a recent report argued against the presence of NO in Leydig cells, proposing instead that this gas was produced by testicular macrophages (52). We therefore thought it of interest to determine whether we would detect changes in NOS and/or NO levels in our models. We show here that indeed, the icv injection of ISO, CRF, or EtOH induced significant increases in TnNOS protein levels as well as testicular NO concentrations. Pretreatment of the animals with the arginine derivative Nwnitro-arginine methylester (L-NAME) at doses known to significantly decrease brain and pituitary NO levels (53, 54) completely prevented increased NO production in the testes. However, L-NAME was unable to restore normal StAR or PBR expression in Leydig cells, or hCG-induced T secretion, in ISO-injected rats. Consequently, the physiological importance of testicular NO in mediating the proposed hypothalamic-testicular inhibitory pathway remains to be further clarified.
Materials and Methods
Animals and surgeries
Adult Sprague Dawley male rats (Harlan Sprague Dawley, Inc., Indianapolis, IN), 65–70 d old at the time of the experiments, were housed under controlled lighting conditions (12 h light, 12 h darkness) with food and water available ad libitum. The icv cannulae were implanted in rats anesthetized with ketamine (100 mg/kg)/acepromazine (4 mg/kg)/xylazine (10 mg/kg) and placed in a stereotaxic device (4). Correct placement of the icv cannulae was verified in coronal sections of the brains after icv injection of dye, and only results from rats with correct placement were included in the statistical analysis. The iv cannulae were inserted under isoflurane anesthesia (4). The animals were individually housed after surgery and allowed to recover for 7–10 d (icv) or 2 d (iv) before experimentation. All protocols were approved by The Salk Institute (La Jolla, CA) Institutional Animal Care and Use Committee.
Experimental protocols
All experiments were carried out in freely moving rats, and each experiment was done at least twice. On the day of the experiment, the animals were removed to a soundproof room and individually housed in opaque buckets no later than 0700 h. The icv cannulae were fitted with connectors made of polyethylene tubing, with injection sites positioned outside of the buckets. The icv treatments were administered at time points listed in the figures (1 μl/10 sec, total volume of 5 μl). The iv cannulae were connected to the injection syringes filled with heparinized saline. After iv injection of hCG (1 U/kg), blood samples (0.3 ml) were taken through the iv cannula in undisturbed rats and immediately replaced with an equivalent volume of apyrogenic isotonic saline. Bloods were drawn into tubes that contained EDTA (10 μl of a 60 mg/ml solution) and placed on ice. They were centrifuged at 4 C, and plasma was stored at –20 C until assayed.
Reagents
Absolute, reagent-grade EtOH (USP, 200 proof) was purchased from Accurate Chemical and Scientific Co. (Shelbyville, KY). We have previously shown that the dose we used (5 μl undiluted EtOH) does not leak to the periphery and does not induce tissue damage within the brain (9, 55). The animals do not exhibit any observable signs of intoxication, and alcohol levels in the cerebrospinal fluid remain virtually undetectable (55). ISO, hCG, and L-NAME were purchased from Sigma Chemical Co. (St. Louis, MO). Rat/human CRF and the LHRH antagonist azaline-B were synthesized by solid-phase methodology (56) and generously provided by Dr. Jean Rivier (The Salk Institute, La Jolla, CA). ISO and CRF were diluted in sterile apyrogenic water for icv injection. Rat StAR antiserum was generously provided by Dr. D. B. Hales (University of Illinois at Chicago, Chicago, IL) and Dr. D. M. Stocco (Texas Tech University, Lubbock, TX). Rat PBR antisera was the generous gift of Dr. V. Papadopoulos (Georgetown University, Washington, DC), whereas P450scc antibodies were generously provided by Dr. W. L. Miller (University of California San Francisco, San Francisco, CA). These antibodies were generated as previously described (26, 57, 58) and used according to the parameters recommended by the corresponding investigators. Collagenase was purchased from Worthington Biochemical Corp. (Lakewood, NJ), and Percoll was purchased from GE Healthcare (Piscataway, NJ). Medium 199 (M199) was purchased from Invitrogen (San Diego, CA).
Leydig cell isolation
The rats were decapitated at the appropriate times after each treatment. Testes were rapidly removed, decapsulated, and cleared of large blood vessels. Samples were dispersed in a collagenase solution (collagenase 0.1% wt/vol, BSA 1.25% wt/vol in M199), washed in M199, suspended in an isotonic Percoll gradient, and centrifuged at 16,000 rpm for 45 min. An equal volume of cells per sample was then pulled off the gradient after centrifugation and washed in heparin dissociation buffer. The purity of Leydig cells obtained by this method is considered to be at least 80% (23, 59, 60). Because of the filtering step used before Percoll gradient, the possible contamination by macrophages is considered to be less than 1% (Hales, D. B., personal communication).
Western blot analysis
Pelleted Leydig cells were suspended in lysis buffer (0.1% SDS in PBS) with Halt protease inhibitor cocktail and MG-132 (Pierce, Rockford, IL), vortexed, briefly sonicated, and centrifuged. Protein concentrations were measured using the Bradford assay for total protein. Equal amounts of protein per sample (75–100 μg) were loaded onto 10% Bis-Tris or 3–8% Tris acetate polyacrylamide gels (Invitrogen) and transferred to nitrocellulose membranes, 0.2 μm pore size (Invitrogen). Blots were incubated separately with 1:500 rat anti-nNOS (Amersham, Piscataway, NJ), 1:5000 rat anti-StAR (Dr. D. B. Hales), 1:3000 human anti-P450scc (Dr. W. L. Miller) (58), or 1:2000 rat anti-PBR (Dr. V. Papadopoulos). Total protein was controlled by measuring levels of the 43-kDa actin with mouse antiactin (Sigma), which showed linear increases over a 25- to 125-μg range (R = 0.923). Western blot detection was performed using a SuperSignal West Pico enhanced chemiluminescent (ECL) kit (Pierce Biotechnology, Rockford, IL), and horseradish peroxidase-conjugated donkey antirabbit from Pierce or sheep antimouse from Amersham Biosciences (Arlington Heights, IL) as secondary antibodies. Semiquantitative analysis of band intensities from protein immunoblots was done using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA) after levels were converted to relative OD. It must be noted that although the active 37-kDa form of StAR is readily visualized in Leydig cells maintained in culture (see for example Ref.32), current procedures for Leydig cell preparation from live rats are often not compatible with the measurement of this form of StAR, which is rapidly processed in the mitochondria (61, 62). However, the direct conversion of the precursor form to the mature 30-kDa form, which can be reliably measured, provides a widely used index of changes in StAR synthesis (62, 63).
In view of the large number of replicates present in most experiments, it was not possible to run all samples under identical conditions. Data were therefore calculated as percent changes vis-a-vis controls. Nevertheless, to demonstrate that the information did not depend on how we presented the data, we also show the raw data for one representative experiment done with icv CRF (see Fig. 2B) because it pertains to groups that were processed under the most closely related conditions.
Assays
Plasma T levels were measured in 50 μl unextracted plasma with a kit from Diagnostic Products Corp. (Los Angeles, CA) (8). The ED20, ED50, and ED80 of this assay are 12.08 ± 1.15, 1.76 ± 0.84, and 0.26 ± 0.04 ng T/ml, respectively. The intra- and interassay coefficients of variation are 2.1 and 4%, respectively. Intratesticular NO levels were measured in isolated Leydig cells using a nitrate/nitrite colorimetric assay (Caymen Chemicals, Ann Arbor, MI), which we have previously described in detail (65, 66). Samples were filtered on Acrodisc 0.2 μM syringe tip filters (Pall, East Hills, NY), and then combined with kit enzymes. Samples were read at 540–550 nm on a BIORAD Lumimark Plus microplate reader. Data were analyzed by MikroWin 2000.
Statistical analysis
Data were analyzed by ANOVA followed by Fisher’s protected least significant difference and Bonferroni/Dunn as a post hoc test. Each value was expressed as the mean ± SEM, and statistical significance was accepted for P < 0.05.
Results
Effect of the icv injection of ISO, EtOH, or CRF on testicular StAR, PBR, and P450scc protein levels (Figs. 1–3)
EtOH, ISO, or CRF was injected icv at doses previously shown to significantly inhibit the T response to hCG (4, 5, 9). Preliminary experiments were first carried out with testes collected over the time courses indicated by these studies, and these data were used to focus on the specific time points illustrated here. Although we show statistical analysis for all time points, we only illustrate immunoblots representative of maximum changes (Figs. 1 and 3) unless protein levels displayed a differential recovery (Fig. 2). In ISO-injected rats, StAR and PBR levels were reduced (P < 0.01) at both the 5- and 30-min time points (Fig. 1). CRF also significantly (P < 0.01) decreased StAR and PBR levels measured 10 min later (Fig. 2, A and B). (For the rationale for presenting results as a function of percent control as well as raw data, see Materials and Methods.) However, PBR levels had returned to control levels by 30 min, a finding that has been consistent. Finally, EtOH decreased PBR (P < 0.01) but not StAR levels 5 min later, whereas values of both proteins were significantly (P < 0.01) lowered at 20 min (Fig. 3). In none of these experiments did P450scc levels exhibit significant changes, which is illustrated for CRF and EtOH in Figs. 2 and 3 (for P450scc response to icv ISO, see Fig. 5 below). Overall, these results indicate that the icv injection of ISO, CRF, or EtOH rapidly, significantly, and consistently decreased the expression of both StAR and PBR measured by Western blotting. Whether these rapid changes in protein levels represent arrested synthesis, increased degradation, or both remains to be determined. Although it is well known that compounds delivered to the brain ventricles readily leak to the periphery (67, 68), we found that the iv injection of amounts of EtOH, ISO, or CRF identical to those administered icv did not alter testicular activity (Rivier, C., unpublished observation). Also, we previously reported that the icv injection of EtOH did not lead to measurable levels of the drug in the circulation (69). Consequently, it is highly unlikely that the changes we report here in terms of StAR and PBR levels result from a peripherally mediated effect of the tested compounds.
Effect of the icv injection of ISO, EtOH, or CRF on Leydig cell TnNOS protein levels and NO levels (Figs. 4 and 5)
The icv injection of ISO, CRF, or EtOH rapidly and significantly (P < 0.01) increased levels of the 125-kDa TnNOS measured 5 and 30 (ISO), 10 and 30 (CRF), or 5 and 20 (EtOH) min later (Fig. 4). Testicular NO levels also significantly (P < 0.05) increased (Fig. 5). In contrast, values for the 150-kDa nNOS isoform showed no significant changes at 10 min (96 ± 35% of control) and inconsistent increases at 30 min (234 ± 55% of control).
Consequence of blocking NOS activity on the effect of ISO on testicular TnNOS, StAR, PBR, and P450scc levels (Fig. 6)
L-NAME was injected according to a regimen (50 mg/kg, sc, 2.5–3 h before icv-injected compounds) previously shown to significantly decrease NOS activity and completely block NO formation in the brain (53, 65). Control rats received the vehicle. In the current experiments, L-NAME similarly interfered with ISO-induced increases in TnNOS levels (Fig. 6A). By contrast, the inhibition of StAR and PBR remained unaffected (Fig. 6B). Finally, P450scc levels were neither decreased by icv-injected ISO nor altered by L-NAME (Fig. 6B).
Consequence of blocking NOS activity on the inhibitory effect of ISO and on hCG-induced T release (Fig. 7)
Even though icv-injected ISO acts on Leydig cell function independently of LH release (4, 6), we considered the possibility that the known influence of NO on the LHRH-LH axis (see for example Refs.71, 72, 73, 74, 75) might introduce a confounding parameter. We therefore ran some experiments in animals pretreated with the LHRH antagonist azaline-B according to a protocol (40 μg/kg, iv, 1 h before L-NAME) that does not influence the inhibitory effect of icv-injected compounds on Leydig cell activity (6, 7). The animals were then injected as described earlier with the sc administration of L-NAME or its vehicle, followed by the central injection of ISO, CRF, or the vehicle. There was no difference between results obtained in rats pretreated or not with azaline-B, and we therefore present data from the latter group. T levels are illustrated as cumulative amounts measured over a 60-min time course after hCG injection. In agreement with a previous report (49), blockade of NO synthesis slightly increased both basal T levels and the T response to hCG (Fig. 7). However, L-NAME did not significantly reverse the inhibitory effect of ISO (percent decrease of the T response to hCG for vehicle/ISO was 59.2 ± 6.0 and for L-NAME/ISO was 58.1 ± 5.7; P > 0.05) or CRF (vehicle/CRF, 42.8 ± 4.6; L-NAME/CRF, 47.6 ± 5.1; P > 0.05) (Fig. 7).
Discussion
The data presented here indicate that the icv injection of the -adrenergic agonist isoproterenol, the stress peptide CRF, or a small amount of alcohol all significantly depressed Leydig cell levels of the steroidogenic proteins StAR and PBR. These results, which extend our previous work on the influence of icv-injected IL-1 on testicular enzymes (13), are interesting for several reasons. First, they provide evidence for concomitant changes in testicular StAR and PBR values in the live male rat, because the only currently available work focuses on levels of these proteins in cultured mouse Leydig tumor cells (32). Our results also provide evidence that these decreases occur very quickly after icv injection of the test compounds but that, interestingly, levels of StAR and PBR returned to control values at different rates. Finally, ISO, CRF, or EtOH did not alter P450scc levels, which indicates that we did not simply measure nonspecific changes in mitochondrial function. It is of interest that icv-injected IL-1 (13) and systemic endotoxemia (19) similarly interfere with testicular steroidogenesis through StAR-dependent mechanisms (PBR was not studied in these experiments) but leave intact the steroidogenic machinery downstream of cholesterol transport.
In view of the current concept that StAR and PBR act in a coordinated manner to transfer cholesterol into mitochondria (28, 29, 76), it was not particularly surprising to observe that levels of both proteins decreased concomitantly. What was more unexpected was the fact that although both StAR and PBR levels remained depressed for at least 30 min after ISO injection, PBR concentrations returned to control values 30 min after CRF administration. It is currently thought that cytoplasmic StAR transfers cholesterol to PBR for import across the membrane (31) while at the same time PBR is required for StAR import into mitochondria (32). Studies done in mouse Leydig tumor cells treated with PBR or StAR oligodeoxynucleotides antisense indicated that the absence of PBR stopped hormone-induced steroidogenesis more quickly than the absence of StAR (32). The authors of this work further proposed that "PBR serves as a gatekeeper in protein and cholesterol import into mitochondria, and StAR serves the role of the hormone-induced activator" (32). Our published (6, 77) as well as unpublished work (C.R.) indicates that CRF significantly depresses the T response to hCG for at least 90 min after its icv administration, thereby encompassing a time when PBR levels are back to control values. This would suggest that once the events controlled by PBR (in particular, cholesterol transfer from the outer to the inner mitochondrial membrane) have taken place, the subsequent inhibition of steroidogenesis depends on other parameters. It is of interest that a similar dissociation between changes in StAR and PBR expression was reported in Leydig cells exposed to hCG despite the continuous release of progesterone (32, 78). Detailed time-related changes in PBR, StAR, P450scc, and other steroidogenic proteins, as well as the respective roles of these entities, therefore need to be evaluated in future experiments focused on our models. At present, we do not know how long ISO and CRF remain in the brain ventricles, what their effective molar concentrations are at the target -adrenergic receptors, and/or how long they retain their activity at the level of these receptors. It is therefore difficult to provide a cogent explanation for their apparent different time course of influence on StAR and PBR.
The rapidity with which icv treatments altered PBR and StAR levels both supports and extends our previous work showing that the icv injection of ISO, for example, significantly interfered with the T response to hCG within 5 min (4). Indeed, the speed of effect of the administration of catecholamines, IL-1, or CRF on Leydig cell activity had provided the original basis for our hypothesis that there was a neural connection between the brain (and in particular the hypothalamus) (4) and the testes (6, 7, 11). We also hypothesized that the intratesticular signal(s) that modulated the influence of this pathway on Leydig cells is required to be released very quickly. This is one of the reasons we were originally attracted to the possibility that this signal might be NO. We show here that, indeed, the icv injection of ISO, CRF, or EtOH significantly up-regulated TnNOS expression in Leydig cells. First, we must note that these findings are in direct contrast to a recent report that Leydig cells are unable to produce NO (52). These investigators proposed that testicular macrophages were the best candidates as a source of NO. At present, the reason for this discrepancy is not clear. However, it should be pointed out that Weissman et al. (52) used a probe directed against brain nNOS, not TnNOS. The latter shows a restricted expression pattern of an nNOS variant in Leydig cells (47), and because we detected only inconsistent and very heterogeneous changes in nNOS in most experiments in which the icv injection of ISO, CRF, or EtOH significantly increased TnNOS levels (see Fig. 4), this may provide at least a partial explanation for the reported failure (52) to observe nNOS in unstimulated Leydig cells. It is also possible that the methods Weissman et al. (52) used to measure NO may not have been sensitive enough to detect basal concentrations of this gas in Leydig cells. Indeed, although we were able to detect the presence of NO itself under both basal and stimulated conditions, changes detected by colorimetry are relatively small, although significant, vis-a-vis those of changes in TnNOS protein levels (see Fig. 5). In this context it is important to remember that NO is very labile and difficult to measure directly (79, 80, 81). Consequently, increases in NOS levels are often considered more reliable indexes of the production of this gas.
Several investigators showed that the addition of large doses of exogenous NO to isolated Leydig cells interferes with steroidogenesis (50, 51, 52). However, despite the ability of stressors such as immobilization and alcohol to increase testicular NO levels, evidence that this gas plays a physiological role in regulating T release remains somewhat ambiguous (40, 42, 82), and the most prudent conclusion may be that at present, the role played by NO may depend on the model. We found that despite the clear up-regulation of the testicular NO machinery by icv-injected ISO, CRF, or EtOH, this phenomenon did not appear to be physiologically relevant because blockade of NO synthesis by the arginine derivative L-NAME did not reverse the inhibitory effect of icv-injected treatments on testicular steroidogenic proteins. There is some in vitro evidence that NO primarily acts on P450scc, rather than StAR (50, 51). However, we show here that in vivo manipulations of NO synthesis did not significantly alter P450scc levels in the testes. Furthermore, the fact that L-NAME was unable to reverse the influence of ISO on the T response to hCG makes it highly unlikely that a putative differential effect of this arginine derivative on StAR vs. P450 is of significance in our model. It is also important to note that we used a dose of L-NAME that we had previously reported effective in nearly totally eliminating NO in various tissues (53, 65). Finally, the method we used for purifying Leydig cells is thought to yield a preparation containing less than 1% macrophages (Hales, D. B., personal communication). Even though macrophages are known to produce NO (see for example Refs.64 and 83, 84, 85, 86), they mostly do so through inducible NOS and are therefore highly unlikely to respond over the time frame of our model. It is equally important to note that L-NAME will block NO release regardless of the source of this gas. Our current understanding of the spatial and temporal dynamics of the spread of NO across tissues (70) suggests that Leydig cells represent likely targets for NO irrespective of its sources within the testes. It therefore seems reasonable to conclude that the lack of influence of L-NAME on T release reflects the fact that this gas does not play a physiological role in our model, rather than the potential inadequacy of the NOS inhibitor to block testicular NO formation.
In conclusion, we have shown that the icv injection of ISO, CRF, or EtOH induced an extremely rapid as well as remarkably specific inhibitory influence on Leydig cell steroidogenesis. These results provide evidence for concomitant changes in the concentrations of Leydig cell StAR and PBR in an in vivo model as well as for the speed at which these changes can take place. The fact that none of the experimental manipulations we used significantly altered P450scc levels also strongly argues for the specificity of these effects on very early events of steroidogenesis. Coupled with our previous reports of the loss of T response to hCG observed in rats receiving these icv treatments (6, 7, 8), the reduction in StAR and PBR levels suggests that these proteins play a critical role in the peripheral modulation of the proposed inhibitory brain-testes pathway (5). Although our results do not provide information regarding potential functional relationships between StAR and PBR, they certainly suggest that, as demonstrated in isolated Leydig cells (31, 32), such is the case. Finally, our studies should help resolve a recent controversy regarding the presence of NO in these cells. We provide here clear evidence that not only do Leydig cells indeed contain the NOS subtype that is specific to the male gonad (47), they also quickly respond to stimulation of the brain-testicular pathway by producing increasing levels of this enzyme. However, although altered steroidogenesis was accompanied by up-regulated activity of the NO machinery, it does not appear to be controlled by this gas. Thus, the intratesticular signaling molecules that mediate the influence of a brain-testicular neural pathway on androgen production remain to be identified.
Acknowledgments
We gratefully acknowledge the generous gifts of h/rCRF and azaline-B from Dr. Jean Rivier (The Salk Institute, La Jolla, CA), StAR antibodies from Dr. D. B. Hales (University of Illinois at Chicago, Chicago, IL) and Dr. D. M. Stocco (Texas Tech University, Lubbock, TX), PBR antibodies from Dr. V. Papadopoulos (Georgetown University, Washington, DC), and P450scc antibodies from Dr. W. L. Miller (University of California San Francisco, San Francisco, CA). We are also indebted to Dr. Dan Selvage for the data shown in Fig. 5, to Amy Blount for expert advice during the course of this work, to Dr. Soon Lee for help with the statistical analysis of the data and the actin standard curve, to Yaira Haas and Cristin Roach for excellent technical assistance, and to Debbie Doan for help in the preparation of the manuscript and the figures.
Footnotes
This work was supported by National Institutes of Health Grant AA-12810.
First Published Online October 20, 2005
Abbreviations: CRF, Corticotropin-releasing factor; EtOH, alcohol; hCG, human chorionic gonadotropin; icv, intracerebroventricular; ISO, isoproterenol; M199, medium 199; nNOS, neuronal nitric oxide synthase; PBR, peripheral-type benzodiazepine receptor; P450scc, cholesterol side-chain cleavage cytochrome P450; StAR, steroidogenic acute regulatory; T, testosterone.
Accepted for publication October 3, 2005.
References
Kalra SP, Kalra PS 1983 Neural regulation of luteinizing hormone secretion in the rat. Endocr Rev 4:311–351
McCann SM, Marubayashi U, Sun HQ, Yu WH 1993 Control of follicle-stimulating hormone and luteinizing hormone release by hypothalamic peptides. Ann NY Acad Sci 687:55–58
Saez J 1994 Leydig cells: endocrine, paracrine and autocrine regulation. Endocr Rev 15:574–626
Selvage D, Rivier C 2003 Importance of the paraventricular nucleus of the hypothalamus as a component of a neural pathway between the brain and the testes that modulates testosterone secretion independently of the pituitary. Endocrinology 144:594–598
Selvage DJ, Rivier C 2004 Leydig cell activity is regulated by a neural pathway between the hypothalamus and the testes that does not include the pituitary. In: Recent research developments in endocrinology. Kerala, India: Transworld Research Network; 97–114
Ogilvie K, Rivier C 1998 The intracerebroventricular injection of interleukin-1 blunts the testosterone response to human chorionic gonadotropin: role of prostaglandin- and adrenergic-dependent pathways. Endocrinology 139:3088–3095
Turnbull AV, Rivier C 1997 Inhibition of gonadotropin-induced testosterone secretion by the intracerebroventricular injection of interleukin-1 in the male rat. Endocrinology 138:1008–1013
Rivier C 1999 Alcohol rapidly lowers plasma testosterone levels in the rat: evidence that a neural brain-gonadal pathway may be important for decreased testicular responsiveness to gonadotropin. Alcohol Clin Exp Res 23:38–45
Selvage D, Lee S, Parsons L, Seo D, Rivier C 2004 A hypothalamic-testicular neural pathway is influenced by brain catecholamines, but not testicular blood flow. Endocrinology 145:1750–1759
Gerendai I, Toth IE, Boldogkoi Z, Medveczky I, Halasz B 2000 Central nervous system structures labelled from the testis using the transsynaptic viral tracing technique. J Neuroendocrinol 12:1087–1095
Lee S, Miselis R, Rivier C 2002 Anatomical and functional evidence for a neural hypothalamic-testicular pathway that is independent of the pituitary. Endocrinology 143:4447–4454
Gerendai I 2004 Supraspinal connections of the reproductive organs: structural and functional aspects. Acta Physiol Hung 91:1–21
Ogilvie K, Hales K, Roberts M, Hales D, Rivier C 1999 The inhibitory effect of intracerebroventricularly injected interleukin 1 on testosterone secretion in the rat: role of steroidogenic acute regulatory protein. Biol Reprod 60:527–533
Krueger R, Orme-Johnson N 1983 Acute adrenocorticotropic hormone stimulation of adrenal corticosteroidogenesis. J Biol Chem 258:10159–10167
Pon L, Hartigan J, Orme-Johnson N 1986 Acute ACTH regulation of adrenal corticosteroid biosynthesis: rapid accumulation of a phosphoprotein. J Biol Chem 261:13309–13316
Lacapere JJ, Papadopoulos V 2003 Peripheral-type benzodiazepine receptor: structure and function of a cholesterol-binding protein in steroid and bile acid biosynthesis. Steroids 68:569–585
Anholt R, DeSouza E, Kuhar M, Snyder S 1985 Depletion of peripheral-type benzodiazepine receptors after hypophysectomy in rat adrenal and testis. Eur J Pharmacol 110:41–46
Stocco DM 1997 A StAR search: implications in controlling steroidogenesis. Biol Reprod 56:328–336
Hales KH, Diemer T, Ginde S, Shankar BK, Roberts M, Bosmann HB, Hales DB 2000 Diametric effects of bacterial endotoxin lipopolysaccharide on adrenal and Leydig cell steroidogenic acute regulatory protein. Endocrinology 141:4000–4012
Bauer M, Bridgham J, Langenau D, Johnson A, Goetz F 2000 Conservation of steroidogenic acute regulatory (StAR) protein structure and expression in vertebrates. Mol Cell Endocrinol 168:119–125
Lin D, Sugawara T, Strauss 3rd JF, Clark BJ, Stocco DM, Saenger P, Rogol A, Miller WL 1995 Role of steroidogenesis acute regulatory protein in adrenal and gonadal steroidogenesis. Science 267:1828–1831
Bose H, Lingappa V, Miller W 2002 Rapid regulation of steroidogenesis by mitochondrial protein import. Nature 417:87–91
Payne AH, Hales DB 2004 Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr Rev 25:947–970
Papadopoulos V 1993 Peripheral-type benzodiazepine/diazepam binding inhibitor receptor: biological role in steroidogenic cell function. Endocr Rev 14:222–240
Zisterer DM, Williams DC 1997 Peripheral-type benzodiazepine receptors. Gen Pharmacol 29:305–314
Papadopoulos V, Amri H, Boujrad N, Cascio C, Culty M, Garnier M, Hardwick M, Li H, Vidic B, Brown AS, Reversa JL, Bernassau JM, Drieu K 1997 Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis. Steroids 62:21–28
Culty M, Li H, Boujrad N, Amri H, Vidic B, Bernassau JM, Reversat JL, Papadopoulos V 1999 In vitro studies on the role of the peripheral-type benzodiazepine receptor in steroidogenesis. J Steroid Biochem Mol Biol 69:123–130
Brown RC, Papadopoulos V 2001 Role of the peripheral-type benzodiazepine receptor in adrenal and brain steroidogenesis. Int Rev Neurobiol 46:117–143
Papadopoulos V 2004 In search of the function of the peripheral-type benzodiazepine receptor. Endocr Res 30:677–684
Liu J, Li H, Papadopoulos V 2003 PAP7, a PBR/PKA-RI-associated protein: a new element in the relay of the hormonal induction of steroidogenesis. J Steroid Biochem Mol Biol 85:275–283
West LA, Horvat RD, Roess DA, Barisas BG, Juengel JL, Niswender GD 2001 Steroidogenic acute regulatory protein and peripheral-type benzodiazepine receptor associate at the mitochondrial membrane. Endocrinology 142:502–505
Hauet T, Yao ZX, Bose HS, Wall CT, Han Z, Li W, Hales DB, Miller WL, Culty M, Papadopoulos V 2005 Peripheral-type benzodiazepine receptor-mediated action of steroidogenic acute regulatory protein on cholesterol entry into Leydig cell mitochondria. Mol Endocrinol 19:540–554
Serra M, Pisu MG, Floris I, Floris S, Cannas E, Mossa A, Trapani G, Latrofa A, Purdy RH, Biggio G 2004 Social isolation increases the response of peripheral benzodiazepine receptors in the rat. Neurochem Int 45:141–148
Zilz A, Li H, Castello R, Papadopoulos V, Widmaier EP 1999 Developmental expression of the peripheral-type benzodiazepine receptor and the advent of steroidogenesis in rat adrenal glands. Endocrinology 140:859–864
Raff H, Hong JJ, Oaks MK, Widmaier EP 2003 Adrenocortical responses to ACTH in neonatal rats: effect of hypoxia from birth on corticosterone, StAR, and PBR. Am J Physiol Regul Integr Comp Physiol 284:R78–R85
Salzmann C, Otis M, Long H, Roberge C, Gallo-Payet N, Walker CD 2004 Inhibition of steroidogenic response to adrenocorticotropin by leptin: implications for the adrenal response to maternal separation in neonatal rats. Endocrinology 145:1810–1822
Lee JJ, Eisenberg P, Papadopoulos V, Wang J, Widmaier EP 2004 Reversible changes in adrenocorticotropin (ACTH)-induced adrenocortical steroidogenesis and expression of the peripheral-type benzodiazepine receptor during the ACTH-insensitive period in young rats. Endocrinology 145:2165–2173
Sridaran R, Philip GH, Li H, Culty M, Liu Z, Stocco DM, Papadopoulos V 1999 GnRH agonist treatment decreases progesterone synthesis, luteal peripheral benzodiazepine receptor mRNA, ligand binding and steroidogenic acute regulatory protein expression during pregnancy. J Mol Endocrinol 22:45–54
Burnett AL, Ricker DD, Chamness SL, Maguire MP, Crone JK, Bredt DS, Snyder SH, Chang TSK 1995 Localization of nitric oxide synthase in the reproductive organs of the male rat. Biol Reprod 52:1–7
Kostic TS, Andric SA, Maric D, Stojilkovic SS, Kovacevic R 1999 Involvement of inducible nitric oxide synthase in stress-impaired testicular steroidogenesis. J Endocrinol 163:409–416
Kostic T, Andric S, Maric D, Kovacevic R 2000 Inhibitory effects of stress-activated nitric oxide on antioxidant enzymes and testicular steroidogenesis. J Steroid Biochem Mol Biol 31:299–306
Shi Q, Hales DB, Emanuele NV, Emanuele MA 1998 Interaction of ethanol and nitric oxide in the hypothalamic-pituitary-gonadal axis in the male rat. Alcohol Clin Exp Res 22:1754–1762
Tatsumi N, Fujisawa M, Kanzaki M, Okuda Y, Okada H, Arakawa S, Kamidono S 1997 Nitric oxide production by cultured rat Leydig cells. Endocrinology 138:994–998
Knowles RG, Moncada S 1994 Nitric oxide synthases in mammals. Biochem J 298:249–258
Andrew PJ, Mayer B 1999 Enzymatic function of nitric oxide synthases. Cardiovasc Res 43:521–531
Wang Y, Goligorsky MS, Lin M, Wilcox JN, Marsden PA 1997 A novel, testis-specific mRNA transcript encoding an NH2-terminal truncated nitric-oxide synthase. J Biol Chem 272:11392–11401
Wang Y, Newton DC, Miller TL, Teichert AM, Phillips MJ, Davidoff MS, Marsden PA 2002 An alternative promoter of the human neuronal nitric oxide synthase gene is expressed specifically in Leydig cells. Am J Pathol 160:369–380
Adams ML, Meyer ER, Sewing BN, Cicero TJ 1994 Effects of nitric oxide-related agents on rat testicular function. J Parmacol Exp Ther 269:230–237
Welch C, Watson ME, Poth M, Hong T, Francis GL 1995 Evidence to suggest nitric oxide is an interstitial regulator of Leydig cell steroidogenesis. Metabolism 44:234–238
Pomerantz DK, Pitelka V 1998 Nitric oxide is a mediator of the inhibitory effect of activated macrophages on production of androgen by the Leydig cell of the mouse. Endocrinology 139:922–931
Del Punta K, Charreau EH, Pignataro OP 1996 Nitric oxide inhibits Leydig cell steroidogenesis. Endocrinology 137:5337–5343
Weissman BA, Niu E, Ge R, Sottas CM, Holmes M, Hutson JC, Hardy MP 2005 Paracrine modulation of androgen synthesis in rat Leydig cells by nitric oxide. J Androl 26:369–378
Uribe R, Lee S, Rivier C 1999 Endotoxin stimulates nitric oxide production in the paraventricular nucleus of the hypothalamus through nitric oxide synthase I: correlation with HPA axis stimulation. Endocrinology 140:5971–5981
Kim CK, Rivier C 1998 Influence of nitric oxide synthase inhibitors on the ACTH and cytokine responses to peripheral immune signals. J Neuroendocrinol 10:353–362
Lee S, Selvage D, Rivier C 2004 Site of action of acute alcohol administration in stimulating the rat hypothalamic-pituitary-adrenal axis: comparison between the effect of systemic and intracerebroventricular injection of this drug on pituitary and hypothalamic responses. Endocrinology 145:4470–4479
Kornreich WD, Galyean R, Hernandez JF, Craig AG, Donaldson CJ, Yamamoto G, Rivier C, Vale W, Rivier J 1992 Alanine series of ovine corticotropin releasing factor (oCRF): a structure-activity relationship study. J Med Chem 35:1870–1876
Hales D, Payne A 1989 Glucocorticoid-mediated repression of P450scc mRNA and de novo synthesis in cultured Leydig cells. Endocrinology 124:2099–2104
Black S, Szklarz G, Harikrishna J, Lin D, Wolf C, Miller W 1993 Regulation of proteins in the cholesterol side-chain cleavage system in JEG-3 and Y-1 cells. Endocrinology 132:539–545
Sam A, Sharma A, Lee L, Hales D, Law W, Ferguson J, Bosmann H 1999 Sepsis produces depression of testosterone and steroidogenic acute regulatory (StAR) protein. Shock 11:298–301
Diemer T, Hales DB, Weidner W 2003 Immune-endocrine interactions and Leydig cell function: the role of cytokines. Andrologia 35:55–63
Strauss J, Kallen C, Christenson L, Watari H, Devoto L, Arakane F, Kiriakidou M, Sugawara T 1999 The steroidogenic acute regulatory protein (StAR): a window into the complexities of intracellular cholesterol trafficking. Recent Prog Horm Res 54:639–694
Granot Z, Geiss-Friendlander R, Melamed-Book N, Eimerl S, Timberg R, Weiss A, Hales K, Hales D, Stocco D, Orly J 2003 Proteolysis of normal and mutated steroidogenic acute regulatory proteins in the mitochondria: the fate of unwanted proteins. Mol Endocrinol 17:2461–2476
Granot Z, Silverman E, Friedlander R, Melamed-Book N, Eimeri S, Timberg R, Hales K, Hales D, Stocco D, Orly J 2002 The life cycle of the steroidogenic acute regulatory (StAR) protein: from transcription through proteolysis. Endocr Rev 28:375–386
Salkowski CA, Detore G, McNally R, van Rooijen N, Vogel SN 1997 Regulation of inducible nitric oxide synthase messenger RNA expression and nitric oxide production by lipopolysaccharide in vivo. J Immunol 158:905–912
Kim C, Rivier C 2000 Nitric oxide and carbon monoxide have a stimulatory role in the hypothalamic-pituitary-adrenal response to physico-emotional stressors in rats. Endocrinology 141:2244–2253
Seo DO, Rivier C 2001 Microinfusion of a nitric oxide donor in discrete brain regions activates the hypothalamic-pituitary-adrenal axis. J Neuroendocrinol 13:925–933
Turnbull A, Rivier C 1998 Intracerebroventricular passive immunization. II. Intracerebroventricular infusion of neuropeptide antisera can inhibit neuropeptide signaling in peripheral tissues. Endocrinology 139:128–136
Turnbull A, Rivier C 1998 Intracerebroventricular passive immunization. I. The effect of intracerebroventricular administration of an antiserum to tumor necrosis factor- on the plasma adrenocorticotropin response to lipopolysaccharide in rats. Endocrinology 139:119–127
Selvage D, Hales D, Rivier C 2004 Comparison between the influence of the systemic and central injection of alcohol on Leydig cell activity. Alcohol Clin Exp Res 28:480–488
Philippides A, Husbands P, O’Shea M 2000 Four-dimensional neuronal signaling by nitric oxide: a computational analysis. J Neurosci 20:1199–1207
Ceccatelli S, Hulting AL, Zhang X, Gustafsson L, Villar M, Hkfelt T 1993 Nitric oxide synthase in the rat anterior pituitary gland and the role of nitric oxide in regulation of luteinizing hormone secretion. Proc Natl Acad Sci USA 90:11292–11296
Aguan K, Mahesh VB, Ping L, Bhat G, Brann DW 1996 Evidence for a physiological role for nitric oxide in the regulation of the LH surge: effect of central administration of antisense oligonucleotides to nitric oxide synthase. Neuroendocrinology 64:449–455
Bhat GK, Mahesh VB, Lamar CA, Ping L, Aguan K, Brann DW 1995 Histochemical localization of nitric oxide neurons in the hypothalamus: association with gonadotropin-releasing hormone neurons and co-localization with N-methyl-D-aspartate receptors. Neuroendocrinology 62:187–197
Moretto M, Lopez FJ, Negro-Vilar A 1993 Nitric oxide regulates luteinizing hormone-releasing hormone secretion. Endocrinology 133:2399–2402
Pinilla L, Gonzalez LC, Tena-Sempere M, Bellido C, Aguilar E 2001 Effects of systemic blockade of nitric oxide synthases on pulsatile LH, prolactin, and GH secretion in adult male rats. Horm Res 55:229–235
Stocco DM, Clark BJ 1996 Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev 17:221–244
Rivier C 2003 Role of pro-inflammatory cytokines in regulating the hypothalamic-pituitary-gonadal axis of the male rat. In: Geenen V, Chrousos G, eds. Immunoendocrinology in health and disease. New York: Marcel Dekker; 107–126
Boujrad N, Vidic B, Papadopoulos V 1996 Acute action of choriogonadotropin on Leydig tumor cells: changes in the topography of the mitochondrial peripheral-type benzodiazepine receptor. Endocrinology 137:5727–5730
Archer S 1993 Measurement of nitric oxide in biological models. FASEB J 7:349–360
Allen BW, Coury Jr LA, Piantadosi CA 2002 Electrochemical detection of physiological nitric oxide: materials and methods. Methods Enzymol 359:125–134
Nussler AK, Bruckner UB, Vogt J, Radermacher P 2002 Measuring end products of nitric oxide in vivo. Methods Enzymol 359:75–83
Shi Q, Emanuele NV, Emanuele MA 1998 Effect of nitric oxide synthase inhibitors on preventing ethanol-induced suppression of the hypothalamic-pituitary-gonadal axis in the male rat. Alcohol Clin Exp Res 22:1763–1770
Stuehr D, Nathan C 1989 Nitric oxide: a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med 169:1543–1555
Li XY, Donaldson K, Macnee W 1995 Nitric oxide production, alveolar macrophages and type II alveolar epithelial cells in response to LPS in vivo and in vitro. Biochem Soc Trans 23:S233
Griscavage JM, Wilk S, Ignarro LJ 1995 Serine and cysteine proteinase inhibitors prevent nitric oxide production by activated macrophages by interfering with transcription of the inducible NO synthase gene. Biochem Biophys Res Commun 215:721–729
Weisz A, Cicatiello L, Esumi H 1996 Regulation of the mouse inducible-type nitric oxide synthase gene promoter by interferon-, bacterial lipopolysaccharide and NG-monomethyl-L-arginine. Biochem J 316:209–215(Melissa Herman and Catherine Rivier)
Abstract
Activation of a neural brain-testicular pathway by the intracerebroventricular injection of the -adrenergic agonist isoproterenol (ISO), the hypothalamic peptide corticotropin-releasing factor (CRF), or alcohol (EtOH) rapidly decreases the testosterone (T) response to human chorionic gonadotropin. To elucidate the intratesticular mechanisms responsible for this phenomenon, we investigated the influence of intracerebroventricular-injected ISO, CRF, or EtOH on levels of the steroidogenic acute regulatory (StAR) protein, the peripheral-type benzodiazepine receptor (PBR), and the cytochrome P450 side-chain cleavage enzyme in semipurified Leydig cells. ISO (10 μg), CRF (5 μg), or EtOH (5 μl of 200 proof, a dose that does not induce neuronal damage nor leaks to the periphery) rapidly decreased StAR and PBR but not cytochrome P450 side-chain cleavage enzyme protein levels. Levels of the variant of the neuronal nitric oxide synthase (nNOS) that is restricted to Leydig cells, TnNOS, significantly increased in response to ISO, CRF, and EtOH over the time course of altered StAR/PBR concentrations. However, pretreatment of the rats with Nwnitro-arginine methylester, which blocked ISO-induced increases in TnNOS, neither restored the T response to human chorionic gonadotropin nor prevented the decreases in StAR and PBR. These results provide evidence of concomitant changes in Leydig cell StAR and PBR levels in live rats. They also indicate that activation of a neural brain-testicular pathway rapidly decreases concentrations of these steroidogenic proteins while up-regulating testicular NO production. However, additional studies are necessary to elucidate the functional role played by this gas in our model.
Introduction
TESTOSTERONE (T) SYNTHESIS in the Leydig cells is traditionally thought to be governed by the release of LH from the pituitary, itself under the control of the hypothalamic peptide LHRH (1, 2, 3). However, certain stressors are known to cause a rapid decrease in T release that occurs without measurable changes in mean LH levels (4). The mechanisms by which this inhibition takes place have been the subject of much speculation, including an altered pattern of pulsatile LH secretion as well as the presence in the circulation and/or the testes of compounds that inhibit steroidogenesis (reviewed in Ref.5). We recently proposed an additional and not necessarily mutually exclusive possibility, namely the existence of an inhibitory brain-testes pathway that is activated by stress-related compounds. This hypothesis was based on our observation that the intracerebroventricular (icv) injection of the -adrenergic agonist isoproterenol (ISO), corticotropin-releasing factor (CRF), or alcohol (EtOH) all rapidly and significantly blunted T secretion induced by the exogenous injection of human chorionic gonadotropin (hCG) (4, 6, 7, 8, 9). Blocking LH release with a potent LHRH antagonist neither mimicked nor influenced the suppressive influence of the icv-administered molecules, thereby indicating that this influence was independent of the pituitary. Normal testicular activity was similarly not restored by prior treatment with CRF antibodies, which did not support the hypothesis that the effect of the proposed inhibitory pathway relied on adrenal steroids (5, 6, 7, 8). Finally, we showed that none of the icv treatments we used altered testicular blood flow (9), thereby eliminating the possibility that the blunted T response measured in these experiments was a result of decreased delivery of gonadotropins to and/or impaired T release from Leydig cells. This pathway was subsequently mapped with the retrograde transneuronal tracer pseudo-rabies virus (10, 11, 12) and shown to include specific areas of the hypothalamus (4, 9). However, the testicular mechanisms through which stimulation of this circuit interferes with T synthesis and release have not yet been elucidated. Our earlier studies had indicated that the icv injection of IL-1 did not alter testicular LH/hCG binding affinity but lowered testicular steroidogenic acute regulatory (StAR) protein levels without suppressing mitochondrial function because the addition of a water-permeable form of cholesterol to decapsulated testes prevented the inhibitory influence of the cytokine (13). In the present work, we investigated the possible roles of three proteins, the StAR protein (14, 15), the peripheral-type benzodiazepine receptor (PBR) (16, 17), and the cholesterol side-chain cleavage cytochrome P450 (P450scc), in response to centrally injected compounds known to decrease the T response to hCG.
StAR is synthesized de novo as a 37-kDa precursor that actively binds cholesterol in the cytosol and is then imported into the mitochondria where it is rapidly cleaved to a 30-kDa mature form (18, 19). This highly conserved protein (20) plays an essential role in steroid hormone synthesis by regulating the transfer of cholesterol, which is the substrate for all steroid hormones, from the outer to the inner mitochondrial membrane where P450scc catalyzes its conversion to pregnenolone (3, 21, 22, 23). Delivery of cholesterol to P450scc is thus considered the true rate-limiting step of steroidogenesis. However, StAR is now known to interact with PBR, an 18-kDa protein located on the outer mitochondrial membrane where it is expressed as a cholesterol transporter and serves as a cholesterol channel (24, 25, 26, 27, 28). Specifically, StAR binds cholesterol in the cytoplasm and transfers it to PBR, a step that is followed by transport of this molecule across the outer to the inner mitochondrial membrane (16, 29, 30). The fact that PBR is required for StAR import into mitochondria (31, 32) thus indicates that both proteins represent essential components of steroidogenesis. Indeed, changes in StAR and/or PBR levels have been associated with related changes in the levels of a variety of steroids in the nervous system (33), the adrenals (34, 35, 36, 37), and the corpus luteum (38). However, to our knowledge, there is no information regarding concurrent responses of gonadal StAR and PBR to stressors delivered to the intact animal.
We show here that icv-injected ISO, CRF, or EtOH all rapidly lowered the testicular levels of both StAR and PBR in paradigms associated with blunted T release. These results then led us to ask whether we could also detect changes in the testicular levels of molecules known to inhibit steroidogenesis that might play a functional role in decreased StAR and PBR levels. Our initial focus was on the unstable gas nitric oxide (NO), which has been reportedly found in testes using various detection methods such as nitrite/nitrate levels, citrulline conversion, and NO synthase (NOS) measurement by histochemical procedure (39, 40). Stressors such as immobilization and proinflammatory cytokines, as well as alcohol, were found to increase testicular NO levels (40, 41, 42), and these increases appear to be related to both constitutive and inducible forms of NO (40, 42, 43). Interestingly, a 125-kDa isoform alternate promoter of the 150-kDa human neuronal NOS, the constitutive enzyme responsible for NO formation (44, 45), was recently found in mice Leydig cells and called T neuronal (n)NOS (46, 47). However, despite much evidence that NO interferes with male sex steroid synthesis (41, 48, 49, 50, 51), a recent report argued against the presence of NO in Leydig cells, proposing instead that this gas was produced by testicular macrophages (52). We therefore thought it of interest to determine whether we would detect changes in NOS and/or NO levels in our models. We show here that indeed, the icv injection of ISO, CRF, or EtOH induced significant increases in TnNOS protein levels as well as testicular NO concentrations. Pretreatment of the animals with the arginine derivative Nwnitro-arginine methylester (L-NAME) at doses known to significantly decrease brain and pituitary NO levels (53, 54) completely prevented increased NO production in the testes. However, L-NAME was unable to restore normal StAR or PBR expression in Leydig cells, or hCG-induced T secretion, in ISO-injected rats. Consequently, the physiological importance of testicular NO in mediating the proposed hypothalamic-testicular inhibitory pathway remains to be further clarified.
Materials and Methods
Animals and surgeries
Adult Sprague Dawley male rats (Harlan Sprague Dawley, Inc., Indianapolis, IN), 65–70 d old at the time of the experiments, were housed under controlled lighting conditions (12 h light, 12 h darkness) with food and water available ad libitum. The icv cannulae were implanted in rats anesthetized with ketamine (100 mg/kg)/acepromazine (4 mg/kg)/xylazine (10 mg/kg) and placed in a stereotaxic device (4). Correct placement of the icv cannulae was verified in coronal sections of the brains after icv injection of dye, and only results from rats with correct placement were included in the statistical analysis. The iv cannulae were inserted under isoflurane anesthesia (4). The animals were individually housed after surgery and allowed to recover for 7–10 d (icv) or 2 d (iv) before experimentation. All protocols were approved by The Salk Institute (La Jolla, CA) Institutional Animal Care and Use Committee.
Experimental protocols
All experiments were carried out in freely moving rats, and each experiment was done at least twice. On the day of the experiment, the animals were removed to a soundproof room and individually housed in opaque buckets no later than 0700 h. The icv cannulae were fitted with connectors made of polyethylene tubing, with injection sites positioned outside of the buckets. The icv treatments were administered at time points listed in the figures (1 μl/10 sec, total volume of 5 μl). The iv cannulae were connected to the injection syringes filled with heparinized saline. After iv injection of hCG (1 U/kg), blood samples (0.3 ml) were taken through the iv cannula in undisturbed rats and immediately replaced with an equivalent volume of apyrogenic isotonic saline. Bloods were drawn into tubes that contained EDTA (10 μl of a 60 mg/ml solution) and placed on ice. They were centrifuged at 4 C, and plasma was stored at –20 C until assayed.
Reagents
Absolute, reagent-grade EtOH (USP, 200 proof) was purchased from Accurate Chemical and Scientific Co. (Shelbyville, KY). We have previously shown that the dose we used (5 μl undiluted EtOH) does not leak to the periphery and does not induce tissue damage within the brain (9, 55). The animals do not exhibit any observable signs of intoxication, and alcohol levels in the cerebrospinal fluid remain virtually undetectable (55). ISO, hCG, and L-NAME were purchased from Sigma Chemical Co. (St. Louis, MO). Rat/human CRF and the LHRH antagonist azaline-B were synthesized by solid-phase methodology (56) and generously provided by Dr. Jean Rivier (The Salk Institute, La Jolla, CA). ISO and CRF were diluted in sterile apyrogenic water for icv injection. Rat StAR antiserum was generously provided by Dr. D. B. Hales (University of Illinois at Chicago, Chicago, IL) and Dr. D. M. Stocco (Texas Tech University, Lubbock, TX). Rat PBR antisera was the generous gift of Dr. V. Papadopoulos (Georgetown University, Washington, DC), whereas P450scc antibodies were generously provided by Dr. W. L. Miller (University of California San Francisco, San Francisco, CA). These antibodies were generated as previously described (26, 57, 58) and used according to the parameters recommended by the corresponding investigators. Collagenase was purchased from Worthington Biochemical Corp. (Lakewood, NJ), and Percoll was purchased from GE Healthcare (Piscataway, NJ). Medium 199 (M199) was purchased from Invitrogen (San Diego, CA).
Leydig cell isolation
The rats were decapitated at the appropriate times after each treatment. Testes were rapidly removed, decapsulated, and cleared of large blood vessels. Samples were dispersed in a collagenase solution (collagenase 0.1% wt/vol, BSA 1.25% wt/vol in M199), washed in M199, suspended in an isotonic Percoll gradient, and centrifuged at 16,000 rpm for 45 min. An equal volume of cells per sample was then pulled off the gradient after centrifugation and washed in heparin dissociation buffer. The purity of Leydig cells obtained by this method is considered to be at least 80% (23, 59, 60). Because of the filtering step used before Percoll gradient, the possible contamination by macrophages is considered to be less than 1% (Hales, D. B., personal communication).
Western blot analysis
Pelleted Leydig cells were suspended in lysis buffer (0.1% SDS in PBS) with Halt protease inhibitor cocktail and MG-132 (Pierce, Rockford, IL), vortexed, briefly sonicated, and centrifuged. Protein concentrations were measured using the Bradford assay for total protein. Equal amounts of protein per sample (75–100 μg) were loaded onto 10% Bis-Tris or 3–8% Tris acetate polyacrylamide gels (Invitrogen) and transferred to nitrocellulose membranes, 0.2 μm pore size (Invitrogen). Blots were incubated separately with 1:500 rat anti-nNOS (Amersham, Piscataway, NJ), 1:5000 rat anti-StAR (Dr. D. B. Hales), 1:3000 human anti-P450scc (Dr. W. L. Miller) (58), or 1:2000 rat anti-PBR (Dr. V. Papadopoulos). Total protein was controlled by measuring levels of the 43-kDa actin with mouse antiactin (Sigma), which showed linear increases over a 25- to 125-μg range (R = 0.923). Western blot detection was performed using a SuperSignal West Pico enhanced chemiluminescent (ECL) kit (Pierce Biotechnology, Rockford, IL), and horseradish peroxidase-conjugated donkey antirabbit from Pierce or sheep antimouse from Amersham Biosciences (Arlington Heights, IL) as secondary antibodies. Semiquantitative analysis of band intensities from protein immunoblots was done using ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA) after levels were converted to relative OD. It must be noted that although the active 37-kDa form of StAR is readily visualized in Leydig cells maintained in culture (see for example Ref.32), current procedures for Leydig cell preparation from live rats are often not compatible with the measurement of this form of StAR, which is rapidly processed in the mitochondria (61, 62). However, the direct conversion of the precursor form to the mature 30-kDa form, which can be reliably measured, provides a widely used index of changes in StAR synthesis (62, 63).
In view of the large number of replicates present in most experiments, it was not possible to run all samples under identical conditions. Data were therefore calculated as percent changes vis-a-vis controls. Nevertheless, to demonstrate that the information did not depend on how we presented the data, we also show the raw data for one representative experiment done with icv CRF (see Fig. 2B) because it pertains to groups that were processed under the most closely related conditions.
Assays
Plasma T levels were measured in 50 μl unextracted plasma with a kit from Diagnostic Products Corp. (Los Angeles, CA) (8). The ED20, ED50, and ED80 of this assay are 12.08 ± 1.15, 1.76 ± 0.84, and 0.26 ± 0.04 ng T/ml, respectively. The intra- and interassay coefficients of variation are 2.1 and 4%, respectively. Intratesticular NO levels were measured in isolated Leydig cells using a nitrate/nitrite colorimetric assay (Caymen Chemicals, Ann Arbor, MI), which we have previously described in detail (65, 66). Samples were filtered on Acrodisc 0.2 μM syringe tip filters (Pall, East Hills, NY), and then combined with kit enzymes. Samples were read at 540–550 nm on a BIORAD Lumimark Plus microplate reader. Data were analyzed by MikroWin 2000.
Statistical analysis
Data were analyzed by ANOVA followed by Fisher’s protected least significant difference and Bonferroni/Dunn as a post hoc test. Each value was expressed as the mean ± SEM, and statistical significance was accepted for P < 0.05.
Results
Effect of the icv injection of ISO, EtOH, or CRF on testicular StAR, PBR, and P450scc protein levels (Figs. 1–3)
EtOH, ISO, or CRF was injected icv at doses previously shown to significantly inhibit the T response to hCG (4, 5, 9). Preliminary experiments were first carried out with testes collected over the time courses indicated by these studies, and these data were used to focus on the specific time points illustrated here. Although we show statistical analysis for all time points, we only illustrate immunoblots representative of maximum changes (Figs. 1 and 3) unless protein levels displayed a differential recovery (Fig. 2). In ISO-injected rats, StAR and PBR levels were reduced (P < 0.01) at both the 5- and 30-min time points (Fig. 1). CRF also significantly (P < 0.01) decreased StAR and PBR levels measured 10 min later (Fig. 2, A and B). (For the rationale for presenting results as a function of percent control as well as raw data, see Materials and Methods.) However, PBR levels had returned to control levels by 30 min, a finding that has been consistent. Finally, EtOH decreased PBR (P < 0.01) but not StAR levels 5 min later, whereas values of both proteins were significantly (P < 0.01) lowered at 20 min (Fig. 3). In none of these experiments did P450scc levels exhibit significant changes, which is illustrated for CRF and EtOH in Figs. 2 and 3 (for P450scc response to icv ISO, see Fig. 5 below). Overall, these results indicate that the icv injection of ISO, CRF, or EtOH rapidly, significantly, and consistently decreased the expression of both StAR and PBR measured by Western blotting. Whether these rapid changes in protein levels represent arrested synthesis, increased degradation, or both remains to be determined. Although it is well known that compounds delivered to the brain ventricles readily leak to the periphery (67, 68), we found that the iv injection of amounts of EtOH, ISO, or CRF identical to those administered icv did not alter testicular activity (Rivier, C., unpublished observation). Also, we previously reported that the icv injection of EtOH did not lead to measurable levels of the drug in the circulation (69). Consequently, it is highly unlikely that the changes we report here in terms of StAR and PBR levels result from a peripherally mediated effect of the tested compounds.
Effect of the icv injection of ISO, EtOH, or CRF on Leydig cell TnNOS protein levels and NO levels (Figs. 4 and 5)
The icv injection of ISO, CRF, or EtOH rapidly and significantly (P < 0.01) increased levels of the 125-kDa TnNOS measured 5 and 30 (ISO), 10 and 30 (CRF), or 5 and 20 (EtOH) min later (Fig. 4). Testicular NO levels also significantly (P < 0.05) increased (Fig. 5). In contrast, values for the 150-kDa nNOS isoform showed no significant changes at 10 min (96 ± 35% of control) and inconsistent increases at 30 min (234 ± 55% of control).
Consequence of blocking NOS activity on the effect of ISO on testicular TnNOS, StAR, PBR, and P450scc levels (Fig. 6)
L-NAME was injected according to a regimen (50 mg/kg, sc, 2.5–3 h before icv-injected compounds) previously shown to significantly decrease NOS activity and completely block NO formation in the brain (53, 65). Control rats received the vehicle. In the current experiments, L-NAME similarly interfered with ISO-induced increases in TnNOS levels (Fig. 6A). By contrast, the inhibition of StAR and PBR remained unaffected (Fig. 6B). Finally, P450scc levels were neither decreased by icv-injected ISO nor altered by L-NAME (Fig. 6B).
Consequence of blocking NOS activity on the inhibitory effect of ISO and on hCG-induced T release (Fig. 7)
Even though icv-injected ISO acts on Leydig cell function independently of LH release (4, 6), we considered the possibility that the known influence of NO on the LHRH-LH axis (see for example Refs.71, 72, 73, 74, 75) might introduce a confounding parameter. We therefore ran some experiments in animals pretreated with the LHRH antagonist azaline-B according to a protocol (40 μg/kg, iv, 1 h before L-NAME) that does not influence the inhibitory effect of icv-injected compounds on Leydig cell activity (6, 7). The animals were then injected as described earlier with the sc administration of L-NAME or its vehicle, followed by the central injection of ISO, CRF, or the vehicle. There was no difference between results obtained in rats pretreated or not with azaline-B, and we therefore present data from the latter group. T levels are illustrated as cumulative amounts measured over a 60-min time course after hCG injection. In agreement with a previous report (49), blockade of NO synthesis slightly increased both basal T levels and the T response to hCG (Fig. 7). However, L-NAME did not significantly reverse the inhibitory effect of ISO (percent decrease of the T response to hCG for vehicle/ISO was 59.2 ± 6.0 and for L-NAME/ISO was 58.1 ± 5.7; P > 0.05) or CRF (vehicle/CRF, 42.8 ± 4.6; L-NAME/CRF, 47.6 ± 5.1; P > 0.05) (Fig. 7).
Discussion
The data presented here indicate that the icv injection of the -adrenergic agonist isoproterenol, the stress peptide CRF, or a small amount of alcohol all significantly depressed Leydig cell levels of the steroidogenic proteins StAR and PBR. These results, which extend our previous work on the influence of icv-injected IL-1 on testicular enzymes (13), are interesting for several reasons. First, they provide evidence for concomitant changes in testicular StAR and PBR values in the live male rat, because the only currently available work focuses on levels of these proteins in cultured mouse Leydig tumor cells (32). Our results also provide evidence that these decreases occur very quickly after icv injection of the test compounds but that, interestingly, levels of StAR and PBR returned to control values at different rates. Finally, ISO, CRF, or EtOH did not alter P450scc levels, which indicates that we did not simply measure nonspecific changes in mitochondrial function. It is of interest that icv-injected IL-1 (13) and systemic endotoxemia (19) similarly interfere with testicular steroidogenesis through StAR-dependent mechanisms (PBR was not studied in these experiments) but leave intact the steroidogenic machinery downstream of cholesterol transport.
In view of the current concept that StAR and PBR act in a coordinated manner to transfer cholesterol into mitochondria (28, 29, 76), it was not particularly surprising to observe that levels of both proteins decreased concomitantly. What was more unexpected was the fact that although both StAR and PBR levels remained depressed for at least 30 min after ISO injection, PBR concentrations returned to control values 30 min after CRF administration. It is currently thought that cytoplasmic StAR transfers cholesterol to PBR for import across the membrane (31) while at the same time PBR is required for StAR import into mitochondria (32). Studies done in mouse Leydig tumor cells treated with PBR or StAR oligodeoxynucleotides antisense indicated that the absence of PBR stopped hormone-induced steroidogenesis more quickly than the absence of StAR (32). The authors of this work further proposed that "PBR serves as a gatekeeper in protein and cholesterol import into mitochondria, and StAR serves the role of the hormone-induced activator" (32). Our published (6, 77) as well as unpublished work (C.R.) indicates that CRF significantly depresses the T response to hCG for at least 90 min after its icv administration, thereby encompassing a time when PBR levels are back to control values. This would suggest that once the events controlled by PBR (in particular, cholesterol transfer from the outer to the inner mitochondrial membrane) have taken place, the subsequent inhibition of steroidogenesis depends on other parameters. It is of interest that a similar dissociation between changes in StAR and PBR expression was reported in Leydig cells exposed to hCG despite the continuous release of progesterone (32, 78). Detailed time-related changes in PBR, StAR, P450scc, and other steroidogenic proteins, as well as the respective roles of these entities, therefore need to be evaluated in future experiments focused on our models. At present, we do not know how long ISO and CRF remain in the brain ventricles, what their effective molar concentrations are at the target -adrenergic receptors, and/or how long they retain their activity at the level of these receptors. It is therefore difficult to provide a cogent explanation for their apparent different time course of influence on StAR and PBR.
The rapidity with which icv treatments altered PBR and StAR levels both supports and extends our previous work showing that the icv injection of ISO, for example, significantly interfered with the T response to hCG within 5 min (4). Indeed, the speed of effect of the administration of catecholamines, IL-1, or CRF on Leydig cell activity had provided the original basis for our hypothesis that there was a neural connection between the brain (and in particular the hypothalamus) (4) and the testes (6, 7, 11). We also hypothesized that the intratesticular signal(s) that modulated the influence of this pathway on Leydig cells is required to be released very quickly. This is one of the reasons we were originally attracted to the possibility that this signal might be NO. We show here that, indeed, the icv injection of ISO, CRF, or EtOH significantly up-regulated TnNOS expression in Leydig cells. First, we must note that these findings are in direct contrast to a recent report that Leydig cells are unable to produce NO (52). These investigators proposed that testicular macrophages were the best candidates as a source of NO. At present, the reason for this discrepancy is not clear. However, it should be pointed out that Weissman et al. (52) used a probe directed against brain nNOS, not TnNOS. The latter shows a restricted expression pattern of an nNOS variant in Leydig cells (47), and because we detected only inconsistent and very heterogeneous changes in nNOS in most experiments in which the icv injection of ISO, CRF, or EtOH significantly increased TnNOS levels (see Fig. 4), this may provide at least a partial explanation for the reported failure (52) to observe nNOS in unstimulated Leydig cells. It is also possible that the methods Weissman et al. (52) used to measure NO may not have been sensitive enough to detect basal concentrations of this gas in Leydig cells. Indeed, although we were able to detect the presence of NO itself under both basal and stimulated conditions, changes detected by colorimetry are relatively small, although significant, vis-a-vis those of changes in TnNOS protein levels (see Fig. 5). In this context it is important to remember that NO is very labile and difficult to measure directly (79, 80, 81). Consequently, increases in NOS levels are often considered more reliable indexes of the production of this gas.
Several investigators showed that the addition of large doses of exogenous NO to isolated Leydig cells interferes with steroidogenesis (50, 51, 52). However, despite the ability of stressors such as immobilization and alcohol to increase testicular NO levels, evidence that this gas plays a physiological role in regulating T release remains somewhat ambiguous (40, 42, 82), and the most prudent conclusion may be that at present, the role played by NO may depend on the model. We found that despite the clear up-regulation of the testicular NO machinery by icv-injected ISO, CRF, or EtOH, this phenomenon did not appear to be physiologically relevant because blockade of NO synthesis by the arginine derivative L-NAME did not reverse the inhibitory effect of icv-injected treatments on testicular steroidogenic proteins. There is some in vitro evidence that NO primarily acts on P450scc, rather than StAR (50, 51). However, we show here that in vivo manipulations of NO synthesis did not significantly alter P450scc levels in the testes. Furthermore, the fact that L-NAME was unable to reverse the influence of ISO on the T response to hCG makes it highly unlikely that a putative differential effect of this arginine derivative on StAR vs. P450 is of significance in our model. It is also important to note that we used a dose of L-NAME that we had previously reported effective in nearly totally eliminating NO in various tissues (53, 65). Finally, the method we used for purifying Leydig cells is thought to yield a preparation containing less than 1% macrophages (Hales, D. B., personal communication). Even though macrophages are known to produce NO (see for example Refs.64 and 83, 84, 85, 86), they mostly do so through inducible NOS and are therefore highly unlikely to respond over the time frame of our model. It is equally important to note that L-NAME will block NO release regardless of the source of this gas. Our current understanding of the spatial and temporal dynamics of the spread of NO across tissues (70) suggests that Leydig cells represent likely targets for NO irrespective of its sources within the testes. It therefore seems reasonable to conclude that the lack of influence of L-NAME on T release reflects the fact that this gas does not play a physiological role in our model, rather than the potential inadequacy of the NOS inhibitor to block testicular NO formation.
In conclusion, we have shown that the icv injection of ISO, CRF, or EtOH induced an extremely rapid as well as remarkably specific inhibitory influence on Leydig cell steroidogenesis. These results provide evidence for concomitant changes in the concentrations of Leydig cell StAR and PBR in an in vivo model as well as for the speed at which these changes can take place. The fact that none of the experimental manipulations we used significantly altered P450scc levels also strongly argues for the specificity of these effects on very early events of steroidogenesis. Coupled with our previous reports of the loss of T response to hCG observed in rats receiving these icv treatments (6, 7, 8), the reduction in StAR and PBR levels suggests that these proteins play a critical role in the peripheral modulation of the proposed inhibitory brain-testes pathway (5). Although our results do not provide information regarding potential functional relationships between StAR and PBR, they certainly suggest that, as demonstrated in isolated Leydig cells (31, 32), such is the case. Finally, our studies should help resolve a recent controversy regarding the presence of NO in these cells. We provide here clear evidence that not only do Leydig cells indeed contain the NOS subtype that is specific to the male gonad (47), they also quickly respond to stimulation of the brain-testicular pathway by producing increasing levels of this enzyme. However, although altered steroidogenesis was accompanied by up-regulated activity of the NO machinery, it does not appear to be controlled by this gas. Thus, the intratesticular signaling molecules that mediate the influence of a brain-testicular neural pathway on androgen production remain to be identified.
Acknowledgments
We gratefully acknowledge the generous gifts of h/rCRF and azaline-B from Dr. Jean Rivier (The Salk Institute, La Jolla, CA), StAR antibodies from Dr. D. B. Hales (University of Illinois at Chicago, Chicago, IL) and Dr. D. M. Stocco (Texas Tech University, Lubbock, TX), PBR antibodies from Dr. V. Papadopoulos (Georgetown University, Washington, DC), and P450scc antibodies from Dr. W. L. Miller (University of California San Francisco, San Francisco, CA). We are also indebted to Dr. Dan Selvage for the data shown in Fig. 5, to Amy Blount for expert advice during the course of this work, to Dr. Soon Lee for help with the statistical analysis of the data and the actin standard curve, to Yaira Haas and Cristin Roach for excellent technical assistance, and to Debbie Doan for help in the preparation of the manuscript and the figures.
Footnotes
This work was supported by National Institutes of Health Grant AA-12810.
First Published Online October 20, 2005
Abbreviations: CRF, Corticotropin-releasing factor; EtOH, alcohol; hCG, human chorionic gonadotropin; icv, intracerebroventricular; ISO, isoproterenol; M199, medium 199; nNOS, neuronal nitric oxide synthase; PBR, peripheral-type benzodiazepine receptor; P450scc, cholesterol side-chain cleavage cytochrome P450; StAR, steroidogenic acute regulatory; T, testosterone.
Accepted for publication October 3, 2005.
References
Kalra SP, Kalra PS 1983 Neural regulation of luteinizing hormone secretion in the rat. Endocr Rev 4:311–351
McCann SM, Marubayashi U, Sun HQ, Yu WH 1993 Control of follicle-stimulating hormone and luteinizing hormone release by hypothalamic peptides. Ann NY Acad Sci 687:55–58
Saez J 1994 Leydig cells: endocrine, paracrine and autocrine regulation. Endocr Rev 15:574–626
Selvage D, Rivier C 2003 Importance of the paraventricular nucleus of the hypothalamus as a component of a neural pathway between the brain and the testes that modulates testosterone secretion independently of the pituitary. Endocrinology 144:594–598
Selvage DJ, Rivier C 2004 Leydig cell activity is regulated by a neural pathway between the hypothalamus and the testes that does not include the pituitary. In: Recent research developments in endocrinology. Kerala, India: Transworld Research Network; 97–114
Ogilvie K, Rivier C 1998 The intracerebroventricular injection of interleukin-1 blunts the testosterone response to human chorionic gonadotropin: role of prostaglandin- and adrenergic-dependent pathways. Endocrinology 139:3088–3095
Turnbull AV, Rivier C 1997 Inhibition of gonadotropin-induced testosterone secretion by the intracerebroventricular injection of interleukin-1 in the male rat. Endocrinology 138:1008–1013
Rivier C 1999 Alcohol rapidly lowers plasma testosterone levels in the rat: evidence that a neural brain-gonadal pathway may be important for decreased testicular responsiveness to gonadotropin. Alcohol Clin Exp Res 23:38–45
Selvage D, Lee S, Parsons L, Seo D, Rivier C 2004 A hypothalamic-testicular neural pathway is influenced by brain catecholamines, but not testicular blood flow. Endocrinology 145:1750–1759
Gerendai I, Toth IE, Boldogkoi Z, Medveczky I, Halasz B 2000 Central nervous system structures labelled from the testis using the transsynaptic viral tracing technique. J Neuroendocrinol 12:1087–1095
Lee S, Miselis R, Rivier C 2002 Anatomical and functional evidence for a neural hypothalamic-testicular pathway that is independent of the pituitary. Endocrinology 143:4447–4454
Gerendai I 2004 Supraspinal connections of the reproductive organs: structural and functional aspects. Acta Physiol Hung 91:1–21
Ogilvie K, Hales K, Roberts M, Hales D, Rivier C 1999 The inhibitory effect of intracerebroventricularly injected interleukin 1 on testosterone secretion in the rat: role of steroidogenic acute regulatory protein. Biol Reprod 60:527–533
Krueger R, Orme-Johnson N 1983 Acute adrenocorticotropic hormone stimulation of adrenal corticosteroidogenesis. J Biol Chem 258:10159–10167
Pon L, Hartigan J, Orme-Johnson N 1986 Acute ACTH regulation of adrenal corticosteroid biosynthesis: rapid accumulation of a phosphoprotein. J Biol Chem 261:13309–13316
Lacapere JJ, Papadopoulos V 2003 Peripheral-type benzodiazepine receptor: structure and function of a cholesterol-binding protein in steroid and bile acid biosynthesis. Steroids 68:569–585
Anholt R, DeSouza E, Kuhar M, Snyder S 1985 Depletion of peripheral-type benzodiazepine receptors after hypophysectomy in rat adrenal and testis. Eur J Pharmacol 110:41–46
Stocco DM 1997 A StAR search: implications in controlling steroidogenesis. Biol Reprod 56:328–336
Hales KH, Diemer T, Ginde S, Shankar BK, Roberts M, Bosmann HB, Hales DB 2000 Diametric effects of bacterial endotoxin lipopolysaccharide on adrenal and Leydig cell steroidogenic acute regulatory protein. Endocrinology 141:4000–4012
Bauer M, Bridgham J, Langenau D, Johnson A, Goetz F 2000 Conservation of steroidogenic acute regulatory (StAR) protein structure and expression in vertebrates. Mol Cell Endocrinol 168:119–125
Lin D, Sugawara T, Strauss 3rd JF, Clark BJ, Stocco DM, Saenger P, Rogol A, Miller WL 1995 Role of steroidogenesis acute regulatory protein in adrenal and gonadal steroidogenesis. Science 267:1828–1831
Bose H, Lingappa V, Miller W 2002 Rapid regulation of steroidogenesis by mitochondrial protein import. Nature 417:87–91
Payne AH, Hales DB 2004 Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocr Rev 25:947–970
Papadopoulos V 1993 Peripheral-type benzodiazepine/diazepam binding inhibitor receptor: biological role in steroidogenic cell function. Endocr Rev 14:222–240
Zisterer DM, Williams DC 1997 Peripheral-type benzodiazepine receptors. Gen Pharmacol 29:305–314
Papadopoulos V, Amri H, Boujrad N, Cascio C, Culty M, Garnier M, Hardwick M, Li H, Vidic B, Brown AS, Reversa JL, Bernassau JM, Drieu K 1997 Peripheral benzodiazepine receptor in cholesterol transport and steroidogenesis. Steroids 62:21–28
Culty M, Li H, Boujrad N, Amri H, Vidic B, Bernassau JM, Reversat JL, Papadopoulos V 1999 In vitro studies on the role of the peripheral-type benzodiazepine receptor in steroidogenesis. J Steroid Biochem Mol Biol 69:123–130
Brown RC, Papadopoulos V 2001 Role of the peripheral-type benzodiazepine receptor in adrenal and brain steroidogenesis. Int Rev Neurobiol 46:117–143
Papadopoulos V 2004 In search of the function of the peripheral-type benzodiazepine receptor. Endocr Res 30:677–684
Liu J, Li H, Papadopoulos V 2003 PAP7, a PBR/PKA-RI-associated protein: a new element in the relay of the hormonal induction of steroidogenesis. J Steroid Biochem Mol Biol 85:275–283
West LA, Horvat RD, Roess DA, Barisas BG, Juengel JL, Niswender GD 2001 Steroidogenic acute regulatory protein and peripheral-type benzodiazepine receptor associate at the mitochondrial membrane. Endocrinology 142:502–505
Hauet T, Yao ZX, Bose HS, Wall CT, Han Z, Li W, Hales DB, Miller WL, Culty M, Papadopoulos V 2005 Peripheral-type benzodiazepine receptor-mediated action of steroidogenic acute regulatory protein on cholesterol entry into Leydig cell mitochondria. Mol Endocrinol 19:540–554
Serra M, Pisu MG, Floris I, Floris S, Cannas E, Mossa A, Trapani G, Latrofa A, Purdy RH, Biggio G 2004 Social isolation increases the response of peripheral benzodiazepine receptors in the rat. Neurochem Int 45:141–148
Zilz A, Li H, Castello R, Papadopoulos V, Widmaier EP 1999 Developmental expression of the peripheral-type benzodiazepine receptor and the advent of steroidogenesis in rat adrenal glands. Endocrinology 140:859–864
Raff H, Hong JJ, Oaks MK, Widmaier EP 2003 Adrenocortical responses to ACTH in neonatal rats: effect of hypoxia from birth on corticosterone, StAR, and PBR. Am J Physiol Regul Integr Comp Physiol 284:R78–R85
Salzmann C, Otis M, Long H, Roberge C, Gallo-Payet N, Walker CD 2004 Inhibition of steroidogenic response to adrenocorticotropin by leptin: implications for the adrenal response to maternal separation in neonatal rats. Endocrinology 145:1810–1822
Lee JJ, Eisenberg P, Papadopoulos V, Wang J, Widmaier EP 2004 Reversible changes in adrenocorticotropin (ACTH)-induced adrenocortical steroidogenesis and expression of the peripheral-type benzodiazepine receptor during the ACTH-insensitive period in young rats. Endocrinology 145:2165–2173
Sridaran R, Philip GH, Li H, Culty M, Liu Z, Stocco DM, Papadopoulos V 1999 GnRH agonist treatment decreases progesterone synthesis, luteal peripheral benzodiazepine receptor mRNA, ligand binding and steroidogenic acute regulatory protein expression during pregnancy. J Mol Endocrinol 22:45–54
Burnett AL, Ricker DD, Chamness SL, Maguire MP, Crone JK, Bredt DS, Snyder SH, Chang TSK 1995 Localization of nitric oxide synthase in the reproductive organs of the male rat. Biol Reprod 52:1–7
Kostic TS, Andric SA, Maric D, Stojilkovic SS, Kovacevic R 1999 Involvement of inducible nitric oxide synthase in stress-impaired testicular steroidogenesis. J Endocrinol 163:409–416
Kostic T, Andric S, Maric D, Kovacevic R 2000 Inhibitory effects of stress-activated nitric oxide on antioxidant enzymes and testicular steroidogenesis. J Steroid Biochem Mol Biol 31:299–306
Shi Q, Hales DB, Emanuele NV, Emanuele MA 1998 Interaction of ethanol and nitric oxide in the hypothalamic-pituitary-gonadal axis in the male rat. Alcohol Clin Exp Res 22:1754–1762
Tatsumi N, Fujisawa M, Kanzaki M, Okuda Y, Okada H, Arakawa S, Kamidono S 1997 Nitric oxide production by cultured rat Leydig cells. Endocrinology 138:994–998
Knowles RG, Moncada S 1994 Nitric oxide synthases in mammals. Biochem J 298:249–258
Andrew PJ, Mayer B 1999 Enzymatic function of nitric oxide synthases. Cardiovasc Res 43:521–531
Wang Y, Goligorsky MS, Lin M, Wilcox JN, Marsden PA 1997 A novel, testis-specific mRNA transcript encoding an NH2-terminal truncated nitric-oxide synthase. J Biol Chem 272:11392–11401
Wang Y, Newton DC, Miller TL, Teichert AM, Phillips MJ, Davidoff MS, Marsden PA 2002 An alternative promoter of the human neuronal nitric oxide synthase gene is expressed specifically in Leydig cells. Am J Pathol 160:369–380
Adams ML, Meyer ER, Sewing BN, Cicero TJ 1994 Effects of nitric oxide-related agents on rat testicular function. J Parmacol Exp Ther 269:230–237
Welch C, Watson ME, Poth M, Hong T, Francis GL 1995 Evidence to suggest nitric oxide is an interstitial regulator of Leydig cell steroidogenesis. Metabolism 44:234–238
Pomerantz DK, Pitelka V 1998 Nitric oxide is a mediator of the inhibitory effect of activated macrophages on production of androgen by the Leydig cell of the mouse. Endocrinology 139:922–931
Del Punta K, Charreau EH, Pignataro OP 1996 Nitric oxide inhibits Leydig cell steroidogenesis. Endocrinology 137:5337–5343
Weissman BA, Niu E, Ge R, Sottas CM, Holmes M, Hutson JC, Hardy MP 2005 Paracrine modulation of androgen synthesis in rat Leydig cells by nitric oxide. J Androl 26:369–378
Uribe R, Lee S, Rivier C 1999 Endotoxin stimulates nitric oxide production in the paraventricular nucleus of the hypothalamus through nitric oxide synthase I: correlation with HPA axis stimulation. Endocrinology 140:5971–5981
Kim CK, Rivier C 1998 Influence of nitric oxide synthase inhibitors on the ACTH and cytokine responses to peripheral immune signals. J Neuroendocrinol 10:353–362
Lee S, Selvage D, Rivier C 2004 Site of action of acute alcohol administration in stimulating the rat hypothalamic-pituitary-adrenal axis: comparison between the effect of systemic and intracerebroventricular injection of this drug on pituitary and hypothalamic responses. Endocrinology 145:4470–4479
Kornreich WD, Galyean R, Hernandez JF, Craig AG, Donaldson CJ, Yamamoto G, Rivier C, Vale W, Rivier J 1992 Alanine series of ovine corticotropin releasing factor (oCRF): a structure-activity relationship study. J Med Chem 35:1870–1876
Hales D, Payne A 1989 Glucocorticoid-mediated repression of P450scc mRNA and de novo synthesis in cultured Leydig cells. Endocrinology 124:2099–2104
Black S, Szklarz G, Harikrishna J, Lin D, Wolf C, Miller W 1993 Regulation of proteins in the cholesterol side-chain cleavage system in JEG-3 and Y-1 cells. Endocrinology 132:539–545
Sam A, Sharma A, Lee L, Hales D, Law W, Ferguson J, Bosmann H 1999 Sepsis produces depression of testosterone and steroidogenic acute regulatory (StAR) protein. Shock 11:298–301
Diemer T, Hales DB, Weidner W 2003 Immune-endocrine interactions and Leydig cell function: the role of cytokines. Andrologia 35:55–63
Strauss J, Kallen C, Christenson L, Watari H, Devoto L, Arakane F, Kiriakidou M, Sugawara T 1999 The steroidogenic acute regulatory protein (StAR): a window into the complexities of intracellular cholesterol trafficking. Recent Prog Horm Res 54:639–694
Granot Z, Geiss-Friendlander R, Melamed-Book N, Eimerl S, Timberg R, Weiss A, Hales K, Hales D, Stocco D, Orly J 2003 Proteolysis of normal and mutated steroidogenic acute regulatory proteins in the mitochondria: the fate of unwanted proteins. Mol Endocrinol 17:2461–2476
Granot Z, Silverman E, Friedlander R, Melamed-Book N, Eimeri S, Timberg R, Hales K, Hales D, Stocco D, Orly J 2002 The life cycle of the steroidogenic acute regulatory (StAR) protein: from transcription through proteolysis. Endocr Rev 28:375–386
Salkowski CA, Detore G, McNally R, van Rooijen N, Vogel SN 1997 Regulation of inducible nitric oxide synthase messenger RNA expression and nitric oxide production by lipopolysaccharide in vivo. J Immunol 158:905–912
Kim C, Rivier C 2000 Nitric oxide and carbon monoxide have a stimulatory role in the hypothalamic-pituitary-adrenal response to physico-emotional stressors in rats. Endocrinology 141:2244–2253
Seo DO, Rivier C 2001 Microinfusion of a nitric oxide donor in discrete brain regions activates the hypothalamic-pituitary-adrenal axis. J Neuroendocrinol 13:925–933
Turnbull A, Rivier C 1998 Intracerebroventricular passive immunization. II. Intracerebroventricular infusion of neuropeptide antisera can inhibit neuropeptide signaling in peripheral tissues. Endocrinology 139:128–136
Turnbull A, Rivier C 1998 Intracerebroventricular passive immunization. I. The effect of intracerebroventricular administration of an antiserum to tumor necrosis factor- on the plasma adrenocorticotropin response to lipopolysaccharide in rats. Endocrinology 139:119–127
Selvage D, Hales D, Rivier C 2004 Comparison between the influence of the systemic and central injection of alcohol on Leydig cell activity. Alcohol Clin Exp Res 28:480–488
Philippides A, Husbands P, O’Shea M 2000 Four-dimensional neuronal signaling by nitric oxide: a computational analysis. J Neurosci 20:1199–1207
Ceccatelli S, Hulting AL, Zhang X, Gustafsson L, Villar M, Hkfelt T 1993 Nitric oxide synthase in the rat anterior pituitary gland and the role of nitric oxide in regulation of luteinizing hormone secretion. Proc Natl Acad Sci USA 90:11292–11296
Aguan K, Mahesh VB, Ping L, Bhat G, Brann DW 1996 Evidence for a physiological role for nitric oxide in the regulation of the LH surge: effect of central administration of antisense oligonucleotides to nitric oxide synthase. Neuroendocrinology 64:449–455
Bhat GK, Mahesh VB, Lamar CA, Ping L, Aguan K, Brann DW 1995 Histochemical localization of nitric oxide neurons in the hypothalamus: association with gonadotropin-releasing hormone neurons and co-localization with N-methyl-D-aspartate receptors. Neuroendocrinology 62:187–197
Moretto M, Lopez FJ, Negro-Vilar A 1993 Nitric oxide regulates luteinizing hormone-releasing hormone secretion. Endocrinology 133:2399–2402
Pinilla L, Gonzalez LC, Tena-Sempere M, Bellido C, Aguilar E 2001 Effects of systemic blockade of nitric oxide synthases on pulsatile LH, prolactin, and GH secretion in adult male rats. Horm Res 55:229–235
Stocco DM, Clark BJ 1996 Regulation of the acute production of steroids in steroidogenic cells. Endocr Rev 17:221–244
Rivier C 2003 Role of pro-inflammatory cytokines in regulating the hypothalamic-pituitary-gonadal axis of the male rat. In: Geenen V, Chrousos G, eds. Immunoendocrinology in health and disease. New York: Marcel Dekker; 107–126
Boujrad N, Vidic B, Papadopoulos V 1996 Acute action of choriogonadotropin on Leydig tumor cells: changes in the topography of the mitochondrial peripheral-type benzodiazepine receptor. Endocrinology 137:5727–5730
Archer S 1993 Measurement of nitric oxide in biological models. FASEB J 7:349–360
Allen BW, Coury Jr LA, Piantadosi CA 2002 Electrochemical detection of physiological nitric oxide: materials and methods. Methods Enzymol 359:125–134
Nussler AK, Bruckner UB, Vogt J, Radermacher P 2002 Measuring end products of nitric oxide in vivo. Methods Enzymol 359:75–83
Shi Q, Emanuele NV, Emanuele MA 1998 Effect of nitric oxide synthase inhibitors on preventing ethanol-induced suppression of the hypothalamic-pituitary-gonadal axis in the male rat. Alcohol Clin Exp Res 22:1763–1770
Stuehr D, Nathan C 1989 Nitric oxide: a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J Exp Med 169:1543–1555
Li XY, Donaldson K, Macnee W 1995 Nitric oxide production, alveolar macrophages and type II alveolar epithelial cells in response to LPS in vivo and in vitro. Biochem Soc Trans 23:S233
Griscavage JM, Wilk S, Ignarro LJ 1995 Serine and cysteine proteinase inhibitors prevent nitric oxide production by activated macrophages by interfering with transcription of the inducible NO synthase gene. Biochem Biophys Res Commun 215:721–729
Weisz A, Cicatiello L, Esumi H 1996 Regulation of the mouse inducible-type nitric oxide synthase gene promoter by interferon-, bacterial lipopolysaccharide and NG-monomethyl-L-arginine. Biochem J 316:209–215(Melissa Herman and Catherine Rivier)