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From the * Department of Pharmacology, Israel
Institute for Biological Research, Ness Ziona, Israel; the
Center for Biomedical Research, Population
Council and the Rockefeller University, New York, New York; the
Division of Neurobiology, Department of
Neurology and Neuroscience, Weill Medical College of Cornell University, New
York, New York; and the Institute of
Neuroendocrinology and the 2nd Affiliated Hospital of Wenzhou Medical College,
Wenzhou, People's Republic of China.
| Correspondence to: Dr Ben Avi Weissman, Department of Pharmacology, IIBR, PO Box 19, Ness Ziona 74100, Israel (e-mail: aviw{at}iibr.gov.il); or Dr Ren-Shan Ge, Population Council, 1230 York Ave, New York, NY 10065 (e-mail: rge{at}popcbr.rockefeller.edu). |
| Received for publication December 29, 2008; accepted for publication March 9, 2009. |
The hormonal changes associated with immobilization stress (IMO) include a
swift increase in corticosterone (CORT) concentration and a decrease in
circulating testosterone (T) levels. There is evidence that the production of
the short-lived neuromodulator nitric oxide (NO) is increased during stress in
various tissues, including the brain. NO also suppresses the biosynthesis of
T. Both the inducible and the neuronal isoforms of NO synthase (iNOS and nNOS,
respectively) have been implicated in this suppression, but the evidence has
not been conclusive. We used adult wild-type (WT) and nNOS knockout male mice
(nNOS–/–) to assess the respective roles of CORT and nNOS-derived
NO in stress mediated inhibition of T production. Animals were assigned to
either basal control or 3-hour IMO groups. No difference in basal plasma and
testicular T levels were observed between WT and nNOS–/–, although
testicular weights of mutant mice were slightly lower compared to WT animals.
The plasma contents of luteinizing hormone (LH) and CORT in unstressed mice of
both genotypes were similar. Exposure to 3 hours of IMO increased plasma CORT
and decreased T concentrations in mice of both genotypes. However, comparable
levels of plasma LH and testicular nitrite and nitrate (NOx), NO stable
metabolites, were detected in control and stressed WT and nNOS–/–
mice. Adrenal concentrations of NOx declined after IMO, but the reduction was
not statistically significant. These findings implicate CORT rather than NO
generated by nNOS in the rapid stress-induced suppression of circulating
T.
Key words: nNOS-null mice, testosterone, glucocorticoid, androgen
One particular aspect to explore is whether nitric oxide synthase (NOS) activity participates in the suppression of T production associated with stress. The presence of neuronal NOS (nNOS) in some areas of the brain and the adrenal gland (Hauser et al, 2005) as well as mammalian testis (Wang et al, 1997) means that NO might have a physiological role in the regulation of regulation of corticosterone (CORT; in rodents) and T homeostasis. Several studies support the view that acute stress (30 minutes to 3 hours) produces a transient increase in nNOS expression in the central nervous system (CNS) (Calza et al, 1993; Kishimoto et al, 1996; de Oliveira et al, 2000; Echeverry et al, 2004; Hori et al, 2005; Okere and Waterhouse, 2006). Furthermore, NO potently changes adrenal gland CORT and aldosterone biosynthesis in vitro (Cymeryng et al, 1998), potently reduces T production in vivo (Adams et al, 1992) and directly suppresses Leydig cells in vitro (Del Punta et al, 1996; Weissman et al, 2005). Thus, there are ample reasons to investigate whether the recruitment of the NO pathway in the adrenal gland during stress is a link between stress stimuli to the brain and suppression of T.
Our recent study on the potential role of inducible NOS (iNOS) in the suppression of T biosynthesis in restrained mice (Weissman et al, 2007) stemmed from reports directly associating the enzyme to hormone levels (eg, Kostic et al, 1999). The observations that IMO produced parallel changes in CORT and T contents in both wild-type (WT) and iNOS-null animals can be explained by the notion that this enzyme is not expressed because of the treatment. Although the decrease in T concentrations was partially attributed to a rapid and direct action of CORT on Leydig cells (Orr and Mann, 1992; Dong et al, 2004), the involvement of nitric oxide (NO) generated by nNOS could not be ruled out. Indeed, numerous reports describe alterations in nNOS expression and activity following IMO stress. Changes in the L-arginine-NO pathway were noted, for instance, in the adrenal gland, indicating a possible effect on CORT release and, thus, modified Leydig cell steroidogenesis. In addition, alterations in nNOS protein and product levels were detected in distinct areas of the mammalian brain such as the paraventricular nucleus (PVN). The localization of a stress-sensitive NO-generating system in this part of the hypothalamus may influence luteinizing hormone (LH) secretion and, in turn, T production. The aim of the present study was to assess the possible involvement of testicular, adrenal, and central nNOS in the stress-induced reduction of Leydig cell steroidogenesis by comparing the physiological responses to IMO in WT and nNOS-deficient (nNOS–/–) mice.
Materials and Methods
Animals
Adult male mice C57BL/6 (25–30-g body weight, n = 35) were purchased
from the Jackson Laboratory (Bar Harbor, Maine). The nNOS–/– mice
(C57BL/6 congenic, n = 32) were obtained from an in-house colony
(Yang et al, 2003), and housed
5 per cage under controlled environmental conditions (temperature 22°C
± 2°C; 12 h light/12 h dark, with lights on from 0600 to 1800
hours). All animals were handled to become adapted for at least 7 days prior
to the beginning of the experiment. The animal protocol was approved by the
Animal Care and Use Committee of Rockefeller University (protocol number
06090).
Immobilization Stress
The animals were placed in wire mesh restrainers (4 x 9 cm in
dimension) as described by McEwen et al
(1995). The procedure
effectively restricted movement. The start of IMO began at 1000 hours and the
control animals were left undisturbed in their cages for the duration of the
experiment and sampled at the same time points. At the end of each stress
period, trunk blood was collected by cardiac puncture, placed in tubes
containing heparin, and centrifuged at 500 x g, and the plasma
supernatants were stored at –20°C until assay. Testes were removed
and stored at –70°C. The overall design was replicated 4 times.
Intratesticular T
Intratesticular T concentrations were measured using the method of Knorr et
al (1970). In brief, frozen
sections of testes (20–60 mg) were homogenized in 5 mL of 70% methanol
using a Polytron homogenizer (30 seconds x 2). The homogenates were
transferred to 15 mL screw cap test tubes. Tracer steroid (1000 cpm of
tritiated T) was added to the homogenate to correct for recovery. The
homogenates were left standing overnight at room temperature. The tubes were
centrifuged at 1800 x g, and the supernatant was aspirated and
dried under nitrogen to remove the methanol. The water layer was extracted
twice with high-performance liquid chromatography–grade diethyl ether.
The ether extracts were then resuspended in 400 µL of radioimmunoassay
(RIA) buffer, and 100 µL was removed for measurement of recovery (the cpm
value in 100 µL x 4 ÷ 1000). The remaining 300 µL was used
for tritium-based T RIA.
Plasma T, LH, and CORT
Plasma T concentrations were measured using a tritium-based RIA as
previously described (Cochran et al,
1981). Plasma LH content was assessed by RIA
(Chandrashekar and Bartke,
1988). Rat LH standards NIDDK-r-LH-I9 and rat LH antibody
NIDDK-anti-rLH-S-11 were obtained through the National Hormone and Pituitary
Program. Rat 125I-labeled LH was obtained through Covance Laboratories
(Vienna, Virginia). IgG antiserum was obtained from ICN Pharmaceuticals (Costa
Mesa, California). Plasma CORT was measured by the RIA procedure of Spencer et
al. (1996), with an anti-CORT
anti B3-163 (Endocrine Sciences, Calabasas, California). Values for interassay
variation of the LH, T, and CORT RIAs were between 4% and 8%. The
sensitivities of the assays for CORT were 10 ng/mL and 10 pg/mL for LH and
T.
Testicular and Adrenal Nitrite and Nitrate
Intratesticular and adrenal nitrite and nitrate (NOx) concentrations were
measured using the Colorimetric Non-enzymatic Assay for Nitric Oxide (Product
No. NB 88; Oxford Biomedical Research, Oxnard, Michigan). In brief, testes and
adrenals were homogenized in 380 and 180 µL of phosphate-buffered saline,
respectively, using a Polytron homogenizer for 30 seconds. Although a single
testicle (100–140 mg) was examined, 1–3 pairs of adrenals
(7–25 mg) were evaluated. Twenty microliters of a 30% solution of
ZnSO4 was added to the homogenates. After 15 minutes' incubation at
room temperature, homogenates were centrifuged at 1000 x g for
20 minutes. Supernatants were transferred to microcentrifuge tubes, and
4–5 cadmium beads were added and tubes shaken overnight. Supernatants
were transferred to microfuge tubes and centrifuged at room temperature for 10
minutes. Samples were kept at –20°C until assay. Duplicate aliquots
(100 µL) were analyzed using the Griess method with sodium nitrite
(0.5–100 µM) to generate a standard curve.
Statistical Analysis
Data were expressed as the mean ± SEM. Statistical evaluation of
plasma, adrenal, and testis parameters was performed by 2-way analysis of
variance (ANOVA) with genotype and treatment as the subclasses. Multiple
comparisons testing was applied to identify significant differences between
groups. Calculations were performed using SPSS software (version 13.0; SPSS,
Chicago, Illinois) or by utilizing Prsm4 (GraphPed Software, San Diego,
California). If ANOVA indicated a significant difference in means, a
Bonferroni statistic was calculated. Two- or one-tailed, nonpaired Student's
t test analyses were used for comparing changes in testicular weights
and T content, respectively.
Results
Testis Weights
Testes of nNOS–/– mice weighed 7.3% less than those of WT
animals: 94.82 ± 1.85 (n = 26) vs 102.3 ± 3.81 (n = 26) mg,
respectively. Following 3 hours of IMO, the testicular weight decreased to
87.26 ± 1.42 (n = 17) and 99.55 ± 3.96 (n = 26), respectively,
and the difference increased to 12.3% and was statistically significant
(P < .02). These results corroborate a previous report describing
the male reproductive phenotype of nNOS–/– mice with exon 6
deletion (Gyurko et al,
2002).
Plasma T Concentration
Figure 1 depicts the effects
of 3 hours of IMO on plasma T concentration. No significant difference was
found between basal plasma T concentrations of WT and congenic
nNOS–/– mice, which were 1.19 ± 0.26 and 0.93 ± 0.17
ng/mL, respectively. Notably, these results mirror our earlier report
concerning WT and iNOS null mice (Weissman
et al, 2007) and resemble data from control and
nNOS–/– mice (Trainor et al,
2007). The reduced plasma T concentrations in both WT and mutant
mice after 3 hours of IMO were significantly lower compared to basal control
animals (F1,61 = 24.9, P < .0001, 2-way
ANOVA). The post-IMO T levels in WT and nNOS–/– mice did not
differ, indicating that genotype was unrelated to the stress response.
|
Plasma CORT Concentration
The effects of 3 hours of IMO on plasma CORT concentrations are depicted in
Figure 2. No significant
difference was observed between basal plasma CORT concentrations of WT and
congenic nNOS–/– mice (Figure
2). Notably, the increase of plasma CORT levels in both WT and
mutant mice subsequent to 3 hours of IMO was significantly higher compared to
basal control animals (F1,52 = 55.86, P <
.0001, 2-way ANOVA). The post-IMO CORT levels in WT and nNOS–/–
mice did not exhibit a statistical difference (P < .05),
indicating that genotype was unrelated to the stress response.
|
Plasma LH Concentrations
There was no statistically significant difference between basal plasma LH
content of WT and nNOS knockout mice, 0.122 ± 0.08 (n = 17) vs 0.312
± 0.2 (n = 13) ng/mL, respectively. These levels of LH are similar to
those previously reported for WT and nNOS–/– mice
(Gyurko et al, 2002). The
application of IMO for 3 hours on mice of both genotypes did not affect their
plasma LH content, which was 0.082 ± 0.048 (n = 16) vs 0.284 ±
0.137 (n = 12) ng/mL, for WT and mutant animals, respectively.
Testicular NOx Levels
The values of testicular NOx concentrations are presented in
Figure 3A. These results are
consistent with published data on the low testicular NOS activity in the rat
and mouse. The values for testicular NOx formation range from less than 0.5 to
650 and 850 fmol/mg protein/min (Burnett et
al, 1995; Tunctan et al,
2002; Hikim et al,
2005, respectively). No statistically significant differences were
detected between all groups (2-way ANOVA;
Figure 3A). Finally, our data
demonstrating the presence of NO metabolites at concentrations between 8 and
13 nmol per gram of tissue are reminiscent to the levels observed in
iNOS–/– mice (Weissman et al,
2007).
|
Adrenal NOx Levels
Adrenals of WT and nNOS–/– mice weighed 7.5 ± 1.1 (n =
9, basal control) vs 7.6 ± 1.1 (n = 9, stressed) and 7.2 ± 0.7
(n = 8, basal control) vs 7.9 ± 1.3 (n = 10, stressed) nmol/g tissue,
respectively. No statistically significant difference could be determined
between these groups (2-way ANOVA). The values of adrenal NOx levels are
depicted in Figure 3B. The
observed 21% decline in NOx content following 3 hours of IMO was not
statistically significant (2-way ANOVA). Our results are in accord with
earlier reports of low NOS activity in testes (<1 pmol/mg protein/min;
Burnett et al, 1995) and higher
NO production in adrenals (1.5 pmol/mg protein/min,
Rettori et al, 2003; 30
pmol/mg protein/min, Cymeryng et al,
2002).
Discussion
The present study examined the outcome of 3 hours of IMO exposure on the levels of T, LH, CORT, and NOx in WT and nNOS–/– mice. Based on the hypothesis that testicular, CNS, and adrenal nNOS and NO may be involved in the control of the decrease in T production that accompanies stress, nNOS–/– animals should exhibit attenuated or absent response. Data presented in this report demonstrate that subsequent to 3 hours of restraint, mice of both genotypes had indistinguishable patterns consisting of lower T coupled with increased plasma CORT concentrations. Additionally, although circulating T content sharply declined in stressed WT and mutant mice, the concentration of testicular NOx, NO-stable metabolites, remained unchanged. Moreover, NOx content in the adrenals and plasma LH levels in all the animals did not reveal any effect of genotype or treatment. Based on these observations, putative testicular, adrenal, or brain NO, produced by nNOS, does not seem to be involved in the reduction of T biosynthesis induced by stress.
In response to IMO, elevation in the expression and activity of NOS in the hypothalamo-pituitary-adrenal axis (HPA) axis have been observed (Calza et al, 1993; Kishimoto et al, 1996; Madrigal et al, 2001; Miyake et al, 2008). Acute IMO (2 hours) induces a significant increase in nNOS mRNA in the medial amygdaloid nucleus, PVN, and other hypothalamic nuclei but not in the hippocampal formation (de Oliveira et al, 2000). Thus, nNOS is present in relevant loci and stress-induced alterations in nNOS and NO levels have been observed. These findings provide a strong basis for the study of nNOS involvement in the restrained-evoked decline of T concentrations. On the other hand, there is a decline in NOx content in the CNS after IMO (Gulati et al, 2006, 2007), as well as evidence that iNOS, the highest NO producer, is inconsequential in IMO-evoked decrease of T production (Sugama et al, 2007; Weissman et al, 2007). Therefore, nNOS–/– animals would be an appropriate model to study the involvement of nNOS in the stress response. This model can provide clearer answers than NOS inhibitors, which are not highly specific blockers, and can affect the HPA system (Rivier and Shen, 1994) as well as corticotropin (ACTH) and CORT concentrations (Giordano et al, 1996).
Mice with targeted disruption of nNOS display grossly normal appearance, locomotor activity, breeding, long-term potentiation, and long-term depression, but do exhibit a large increase in their aggressive behavior and excess, inappropriate sexual behavior (Nelson et al, 1995). The neurons normally expressing NOS appear intact, and the mutant nNOS mice are without evident histopathological abnormalities in the CNS (Huang et al, 1993). Investigators have also shown that nNOS–/– mice do not differ from WT animals in their plasma ACTH (Bernstein et al, 1998), LH level (Gyurko et al, 2002), CORT (Greenberg et al, 1999; Salchner et al, 2004; Orlando et al, 2007), and basal plasma T concentrations (Kriegsfeld et al, 1997; Gyurko et al, 2002; Trainor et al, 2007). Thus the endocrine profiles of nNOS–/– mice appear to validate the use of this model in our study.
Given the potent inhibitory action of NO on Leydig cell steroidogenesis (Del Punta et al, 1996; Weissman et al, 2005), and the presence of nNOS proteins in testis (Wang et al, 1997), an increase in the secretion of T in nNOS–/– mice was expected. However, both basal plasma T concentrations and the stress-induced decline in T levels were similar in WT and iNOS–/– mice. Our measurements of basal plasma T concentrations, intratesticular NOx concentrations, and plasma LH content in nNOS–/– mice were all consistent in magnitude with the published reports mentioned earlier; therefore, we expect that if the mutant mice responded differently than WT mice, we would have been able to show differences.
nNOS mRNA and proteins are found in rat adrenals (Afework et al, 1992; Tanaka and Chiba, 1996; Tsuchiya et al, 1996; Lai et al, 2006), and both have been reported to increase in the adrenals in response to IMO (Kishimoto et al, 1996; Tsuchiya et al, 1996). In a recent report, the rapid, ACTH-induced release of CORT from the adrenal was shown to be mediated by NO (Mohn et al, 2005). Because NO has a dramatic influence on steroidogenesis, the omission of nNOS from the adrenal gland is expected to alter CORT secretion. Our investigation revealed a similar NO production level in all the adrenal glands examined (Figure 3B) and comparable plasma CORT contents (Figure 2). Our basal measurements corroborate published studies (Greenberg et al, 1999; Salchner et al, 2004; Orlando et al, 2007). Our data demonstrating a marked elevation in CORT levels after 3 hours of IMO are in agreement with recent records indicating parallel alterations following swim stress (Salchner et al, 2004; Orlando et al, 2007). Therefore, our measurements and magnitude of changes are consistent with published studies.
The dynamics of stress-induced changes in T, CORT, and LH levels in rats (Orr and Mann, 1992) and mice (Dong et al, 2004) have been investigated. It has been established that LH remains unchanged whereas CORT levels increase in stressed males starting at 15 minutes, and reach a higher plateau by 1 hour. In the same period, both plasma and testicular T concentrations decreased in stressed animals starting from 30 minutes post-IMO. Notably, in vitro CORT treatment of Leydig cells reduced intracellular cyclic adenosine monophosphate content by 15 minutes and T production by 30 minutes. It was concluded that the rapid changes in T result from a suppression of its biosynthesis by glucocorticoid through a nongenomic mechanism involving a direct inhibitory action of CORT on Leydig cells. However, Orr and Mann (1992) and Dong et al (2004) showed partial reversal of the inhibitory effect of stress on plasma T levels by in vivo pretreatment with RU486; therefore, glucocorticoids are probably not the only mediator of stress-induced inhibition of T. Thus, other candidates such as NO should be considered, especially when its generating machinery (ie, NO synthases) was localized to brain, adrenal, testis and Leydig cells.
iNOS, an enzyme producing large quantities of NO, was proposed as a likely source of a potent T biosynthesis suppressor. In fact, several studies recorded increased expression of iNOS following restraint stress (eg, Kostic et al, 1999). Nevertheless, in a recent report we demonstrated that mice lacking iNOS do not differ from congenic WT animals in their response to IMO (Weissman et al, 2007). Our current results show that the constitutive enzyme nNOS, which has been localized to critical loci relevant to CORT and T steroidogenesis, is inconsequential in the process leading from stress to reduced plasma T concentrations. The finding showing that NO produced by nNOS did not regulate NO concentrations in testis and adrenal and had no effect on circulating LH content adds a novel perspective to the issue of NO participation in the control of T secretion. Based on the above observations and because NO is a powerful inhibitor of T production, the potential role of another source of NO, namely, endothelial NOS, should be considered.
Footnotes
Supported by National Institutes of Health grants HD050570 (R.S.G.) and HL018974 and NS 34179 (C.I. and P.Z.).
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